Drug target site within gp120 of HIV

ABSTRACT

The present invention relates to a method of designing an inhibitor of the binding of HIV (human immunodeficiency virus) glycoprotein (gp)120 to a CD4-receptor or to the integrin alpha4 beta7 (a4b7). The inhibitor interacts with at least two amino acid residues comprised in six motifs within the 3-dimensional structure of gp120. Also provided are compounds, pharmaceutical compositions thereof and uses thereof in the development of an inhibitor of the binding of a HIV gp120 to a CD4-receptor or an integrin alpha4 beta7 (a4b7). The inhibitors are useful for the prevention or treatment of an HIV infection and/or diseases associated with an HIV infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 U.S. National Stage Entry ofInternational Application No. PCT/EP2011/068924 filed Oct. 27, 2011,which claims the benefit of priority of European Application No.10014037.5 filed Oct. 27, 2010, the contents of which are eachincorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inASCII format. The Sequence Listing is provided as a file entitled2052.1000US371 SL.txt created on Dec. 10, 2015 which is 130,916 bytes insize. The information in electronic format of the sequence listing isincorporated herein by reference in its entirety.

DETAILED DESCRIPTION

The present invention relates to a method of designing an inhibitor ofthe binding of HIV (human immunodeficiency virus) glycoprotein (gp)120to a CD4-receptor or to the integrin alpha4 beta7 (a4b7), the methodcomprising the molecular modelling of a compound such that the modelledcompound interacts in silico with at least two amino acid residuescomprised in six motifs within the 3-dimensional structure of said gp120of HIV or within a peptidomimetic reflecting the 3-dimensional structureof said gp120 of HIV, wherein said interaction between said at least twoamino acid residues and said compound is characterized by an interatomicdistance of less than 8 Angströms, wherein a first motif of said sixmotifs comprises the amino acid sequence DIISLWDQSLKPCVKLT (SEQ ID NO:3) or a variant thereof; wherein a second motif of said six motifscomprises the amino acid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ IDNO: 4) or a variant thereof; wherein a third motif of said six motifscomprises the amino acid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5)or a variant thereof; wherein a fourth motif of said six motifscomprises the amino acid sequence CPKISFEP (SEQ ID NO: 6) or a variantthereof; wherein a fifth motif of said six motifs comprises the aminoacid sequence FRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variantthereof; and wherein a sixth motif of said six motifs comprises theamino acid sequence CSS or a variant thereof, said gp120 of HIVcomprising or consisting of (i) the sequence of SEQ ID NO: 2; (ii) thesequence encoded by the sequence of SEQ ID NO: 1; (iii) a sequence beingat least 50% identical to the sequence of SEQ ID NO: 2 or to thesequence encoded by the sequence of SEQ ID NO: 1; or (iv) a sequenceencoded by a sequence being at least 50% identical to the sequence ofSEQ ID NO: 1, wherein said sequence of (iii) or (iv) comprises orencodes said motifs or variants thereof. Also, the invention relates toa method of identifying an inhibitor of the binding of HIV gp120 to CD4or the integrin a4b7, compounds that inhibit the binding of HIV gp120 toCD4 or the integrin a4b7, a method of decreasing thermal motion of atunnel within HIV gp120, compounds for use in preventing or treating aHIV-infection and/or a disease associated with a HIV-infection and apharmaceutical composition comprising any of the above compounds orinhibitors. Finally, the invention also relates to a tunnel withingp120.

In this specification, a number of documents including manufacturer'smanuals is cited. The disclosure of these documents, while notconsidered relevant for the patentability of this invention, is herewithincorporated by reference in its entirety. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

Human immunodeficiency virus (HIV) is a retrovirus belonging to theprimate lentiviruses that can lead upon successful infection to acondition termed acquired immunodeficiency syndrome (AIDS).

Said condition is characterized in that the immune system begins to failand therefore the patient's body becomes increasingly susceptible tosecondary and/or recurring infections. The infection with HIV occurs by,e.g., transfer of blood, semen, vaginal fluid and also breast milk. Dueto the presence of unbound infectious virus particles in body fluids therate of infection is high. In particular, sexual intercourse andtransmission from infected mothers to their babies as well as feedingwith breast milk account for a majority of new HIV cases.

Since becoming a pandemic in the 1980's HIV has received much attentionboth in the general public as well as in the scientific community. TheWorld Health Organization (WHO) and the Joint United Nations Programmeon HIV/AIDS (UNAIDS) have recently estimated that about 25 millionpeople have died due to AIDS since 1981 making it one of the mostdestructive pandemics in history. This can be linked back to the uniqueway of cellular infection, manifestation and persistence of theretrovirus in the body which has not yet been found to be successfullytreatable.

Presently, treatment of HIV infected patients relies on combinationtherapies such as, e.g., highly active antiretroviral therapy (HAART),that may be expensive, cause serious drug-related side effects and maygive rise to resistant HIV strains after prolonged progression of thetherapy. Conventional combination therapies comprise nucleoside-analoguereverse transcriptase inhibitors (NARTIs or NRTIs), nonnucleoside-analogue reverse transcriptase inhibitors (NNRTIs) and/orprotease inhibitors.

In addition to reverse transcriptase and protease inhibitors,therapeutic drugs for the treatment or prevention of HIV-relateddiseases have been and continue to be developed which interfere with theprocess of binding and entry of HIV into its target cells. The processof HI-viral entry into a target cells represents the first step in theviral infection circle. It is characterized by a complex series ofevents that are initiated through the binding of the viral surfaceglycoproteins to specific receptor molecules on the cell's outermembrane. This interaction is thought to trigger a conformational changein the viral glycoprotein, which then mediates fusion of the lipidbilayers of the cell and viral membranes and allows the genetic materialof the virus to be introduced into the host-cell cytoplasm.

A more detailed view shows that CD4 is the primary receptor for HIVwhich is a 60 kD molecule on the surface of certain immune cells suchas, e.g., T lymphocytes, cells of the monocyte/macrophage lineage, ordendritic, antigen-presenting cells (Weiss, R. A. (1993), TheRetroviridae, 2nd edition (ed. J. A. Levy), pp. 1-108. Plenum Press, NewYork), and is endogenously involved in T-cell activation (Sweet et al.(1991), Curr. Opin. Biotechnol. 2: 622-633). The virus enters CD4+ cellsand after successful amplification and budding of progeny virusparticles lyses the infected CD4+ cells. Hence, a hallmark of acquiredimmunodeficiency syndrome (AIDS) is the depletion of CD4+ cells. Thebinding of HIV to CD4+ cells involves the formation of a stable complexbetween CD4 and gp120, the glycoprotein exposed on the envelope of HIVthat mediates binding and subsequent entry into the host cell. CD4 hasshown to be necessary and sufficient for efficient HIV attachment totarget cells. Nevertheless, its presence alone is not sufficient forviral entry and the importance of secondary/fusion receptors couldsubsequently be established that mediate the fusion of the virusparticle and the target cell. This requirement of the presence of asecondary/fusion receptor appears to be so far unique to primatelentiviruses. Several studies identified the CXCR4 and the CCR5 receptorwhich have been shown to mediate the fusion of virus particles withdifferent tropisms and the respective target cell. The CXCR4 receptorseems to be specific for T-cell tropic HIV strains whereas the CCR5receptor seems to be specific for M-tropic strains.

In detail, HIV enters macrophages and CD4+ T cells by the adsorption ofglycoproteins on the target cell followed by fusion of the viralenvelope with the cell membrane and the release of the HIV capsid intothe cell (Chan D et Kim P, Cell 93 (5): 681-4 (1998); Wyatt R etSodroski J, Science 280 (5371): 1884-8 (1998). The first step in fusioninvolves the high-affinity attachment of the CD4 binding domains ofgp120 to CD4. Once gp120 is bound to CD4, the envelope complex undergoesa profound conformational change, exposing the chemokine binding domainsof gp120 and allowing them to interact with the target chemokinereceptor (generally either CCR5 or CXCR4, but others are known tointeract). This results in a more stable two-pronged attachment, whichallows the N-terminal fusion peptide gp41 to penetrate the cellmembrane.

Thus, the gp120/CD4 interaction in connection with the subsequentinteraction with the above-identified coreceptors CXCR4 and CCR5provides a potential target for intervention in HIV infections. A numberof antibodies and small molecules have been developed as blockers orinhibitors of the gp120/CD4 binding by interacting with either gp120 orCD4 (Vermeire et al. (2006), Curr. Med. Chem., 13, 731). Common blockersor inhibitors include but are not limited to antisense molecules,antibodies, antagonists, traps, and their derivatives. However, so farnone of these approaches has led to a clinically approved drug.Importantly none of these approaches is designed to target theconformational change undergone by gp120 after binding to CD4. Inparticular, compounds that are shown to interact with binding sites onthe surface of gp120 next to the natural binding site for CD4 could notbe shown to inhibit said conformational change (Kong et al., Biochimicaet Biophysica Acta—Proteins & Proteomics, Elsevier, Netherlands, vol.1764, no. 4, April 2006, 766-772, ISSN:1570-9639; Berchanski et al.,Biochimica et Biophysica Acta—Biomembranes, Netherlands, vol. 1768, no.9, September 2007, 2107-2119, ISSN:1570-9639).

Recently, a further receptor was demonstrated to be critically involvedin the primary infection of CD4+ cells (Arthos et al., NatureImmunology, vol. 9, no. 3 (2008)). It was shown that the HIV envelopeprotein gp120 bound to and signalled by means of integrin alpha4 beta7on CD4+ T lymphocytes. Further, it was shown that gp120 rapidlyactivated LFA-1, an integrin that facilitates HIV infection, on CD4+ Tcells in an alph4 beta7-dependent way. Functioning principally as ahoming receptor, alpha4 beta7 mediates the migration of leukocytes to anretention of leukocytes in the lamina propria of the gut. Thus, in thetissue where HIV preferentially replicates, its envelope interactsdirectly with an adhesion receptor that is specifically linked to thefunction of CD4+ T cells in that tissue.

As evidenced by the above discussion, the efforts to identify anddevelop more efficient drugs and therapies to successfully address theincreasing rate of new HIV infections, of progression to AIDS and theincreasing death toll linked to the latter are intense and everincreasing in view of the rapidly growing knowledge of HIV and itsinteraction with the human host. Despite said efforts there is still noreported success of therapeutic strategies and their technicalimplementation to successfully prevent or to treat HIV infection.

The technical problem underlying the present invention was to identifyalternative and/or improved means and methods for the prevention ortreatment of an HIV infection and/or diseases associated with an HIVinfection. The solution to this technical problem is achieved byproviding the embodiments characterized in the claims.

Accordingly, the present invention relates in a first embodiment to amethod of designing an inhibitor of the binding of HIV (humanimmunodeficiency virus) glycoprotein (gp)120 to a CD4-receptor or to theintegrin alpha4 beta7 (a4b7), the method comprising the molecularmodelling of a compound such that the modelled compound interacts,preferably in silico, with at least two amino acid residues comprised insix motifs within the 3-dimensional structure of said gp120 of HIV orwithin a peptidomimetic reflecting the 3-dimensional structure of saidgp120 of HIV, wherein said interaction between said at least two aminoacid residues and said compound is characterized by an interatomicdistance of less than 8 Angströms, wherein a first motif of said sixmotifs comprises the amino acid sequence DIISLWDQSLKPCVKLT (SEQ ID NO:3) or a variant thereof; wherein a second motif of said six motifscomprises the amino acid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ IDNO: 4) or a variant thereof; wherein a third motif of said six motifscomprises the amino acid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5)or a variant thereof; wherein a fourth motif of said six motifscomprises the amino acid sequence CPKISFEP (SEQ ID NO: 6) or a variantthereof; wherein a fifth motif of said six motifs comprises the aminoacid sequence FRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variantthereof; and wherein a sixth motif of said six motifs comprises theamino acid sequence CSS or a variant thereof, said gp120 of HIVcomprising or consisting of (i) the sequence of SEQ ID NO: 2; (ii) thesequence encoded by the sequence of SEQ ID NO: 1; (iii) a sequence beingat least 50% identical to the sequence of SEQ ID NO: 2 or to thesequence encoded by the sequence of SEQ ID NO: 1; or (iv) a sequenceencoded by a sequence being at least 50% identical to the sequence ofSEQ ID NO: 1, wherein said sequence of (i), (ii), (iii) or (iv)comprises or encodes said motifs or variants thereof.

In accordance with the present invention, the six motifs form a tunnelwithin the 3-dimensional structure of gp120. The term “tunnel” is usedaccording to its well-known meaning and in accordance with the inventionrefers to an elongate hollow or water-filled space with an opening oneither side within the 3-dimensional structure of gp120. The tunnel'sshape is dictated by the position of said six motifs in relation to eachother within said 3-dimensional structure of gp120. As will beunderstood by the skilled person, the six motifs form the (inner) wallsof the tunnel. Hence, any modelled compound or test compound orinhibitor according to the invention interacts with the amino acidresidues comprised in the six motifs making up the walls of the tunnelas contact/interaction sites. As will be explained herein below, themotifs may vary in sequence and length which does, however, not affecttheir capability of forming said tunnel. The above is applicable for allembodiments of the invention. The tunnel may also be fragmented todescribe shorter versions of said tunnel. For example, a shorter versionof the tunnel is made up only of the amino acid residues IISLWDQSLK ofthe first motif (residues 2-11 of SEQ ID NO: 3); IRPVVSTQLLLN of thesecond motif (residues 11-22 of SEQ ID NO: 4); VMHSFNCGGEFFYC of thethird motif (residues 8-21 of SEQ ID NO: 5); CPKISFEP of the fourthmotif (SEQ ID NO: 6); GGDMR of the fifth motif (residues 5-9 of SEQ IDNO: 7); and CSS of the sixth motif. It is understood in accordance withthe invention that the definition of a shorter version of the tunnel canreplace the definition of the tunnel with six motifs throughout theapplication.

The tunnel extends through gp120 from the site where gp120 makes contactwith CD4 (specifically CD4's Phe43 residue, i.e. the binding site ofCD4), i.e. a depression formed by the interface of the outer domain withthe inner domain and the bridging sheet of gp120, towards a site that ison the back of said CD4 binding site, where two glycosylated asparaginresidues at position 120 and 300 in SEQ ID NO: 2 are found, wherein thelatter sites are each on the surface of gp120. Also, said tunnel opensand partially closes as a consequence of thermal motion.

In a further embodiment, the invention relates to a method of designingan inhibitor of the binding of HIV (human immunodeficiency virus)glycoprotein (gp)120 to a CD4-receptor or to the integrin alpha4 beta?(a4b7), the method comprising the molecular modelling of a compound suchthat the modelled compound interacts, preferably in silico, with atleast two amino acid residues comprised in six motifs forming a tunnelwithin the 3-dimensional structure of said gp120 of HIV or within apeptidomimetic reflecting the 3-dimensional structure of said gp120 ofHIV, wherein said interaction between said at least two amino acidresidues and said compound is characterized by an interatomic distanceof less than 8 Angströms, wherein a first motif of said six motifscomprises the amino acid sequence DIISLWDQSLKPCVKLT (SEQ ID NO: 3) or avariant thereof; wherein a second motif of said six motifs comprises theamino acid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ ID NO: 4) or avariant thereof; wherein a third motif of said six motifs comprises theamino acid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5) or a variantthereof; wherein a fourth motif of said six motifs comprises the aminoacid sequence CPKISFEP (SEQ ID NO: 6) or a variant thereof; wherein afifth motif of said six motifs comprises the amino acid sequenceFRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variant thereof; and whereina sixth motif of said six motifs comprises the amino acid sequence CSSor a variant thereof, said gp120 of HIV comprising or consisting of (i)the sequence of SEQ ID NO: 2; (ii) the sequence encoded by the sequenceof SEQ ID NO: 1; (iii) a sequence being at least 50% identical to thesequence of SEQ ID NO: 2 or to the sequence encoded by the sequence ofSEQ ID NO: 1; or (iv) a sequence encoded by a sequence being at least50% identical to the sequence of SEQ ID NO: 1, wherein said sequence of(i), (ii), (iii) or (iv) comprises or encodes said motifs or variantsthereof. The preferred embodiments of the main embodiment describedherein below are also preferred embodiments with regard to thisembodiment.

An interaction with at least two amino acid residues comprised in sixmotifs within the 3-dimensional structure of said gp120 of HIV or withina peptidomimetic reflecting the 3-dimensional structure of said gp120 ofHIV is indicative that an inhibitor of the binding of HIV (humanimmunodeficiency virus) glycoprotein (gp)120 to a CD4-receptor and/or tothe integrin alpha4 beta? (a4b7) has been designed.

It is understood that a compound modeled in accordance with the methodof the invention functions as an inhibitor of said binding of gp120 toCD4 and/or integrin a4b7. Nevertheless, the method of the invention maycomprise the further step (after modeling is completed) of providing themodeled compound; bringing it into contact with a gp120 (isolated or aspart of a HI virus) and determining whether binding occurs.Corresponding experimental setups are well-known in the art and can beeasily devised by a person skilled in the art. Exemplary methods areshown in the example section (Examples 2 and 3).

The term “designing” refers to devising an inhibitor, preferably by insilico methods, i.e. computer-implemented methods. As regards the insilico methods for designing inhibitors, these are commonly referred toas molecular modeling. Particularly envisaged for the present inventionare molecular modeling tools which are also referred to as ligandconstruction tools. Such methods for rational drug design typically takeinto account properties including shape, charge distribution, thedistribution of hydrophobic groups, ionic groups and groups capable offorming hydrogen bonds at a site of interest of the protein moleculeunder consideration. Using this information, that can be derived fromthe high resolution structure of proteins and protein-ligand complexes,these methods either suggest improvements to existing molecules,construct new molecules on their own that are expected to have goodbinding affinity, screen through virtual compound libraries for suchmolecules or fragments thereof, or otherwise support interactive designof new drug compounds in silico. Typically, ligand construction makesuse of dedicated software and involves interactive sessions in front ofa computer display of the three-dimensional structure of the targetmolecule, i.e., gp120, and of candidate molecules or fragments thereof.Suitable software packages are known in the art and include Chemoffice(CambridgeSoft Corporation), CNS (Acta Cryst. D54, 905-921), CCP4 (ActaCryst. D50, 760-763), ADF (Computational Chemistry, David Young,Wiley-Interscience, 2001. Appendix A. A.2.1 p. 332) and Gold (G. Jones,P. Willett and R. C. Glen, J. Mol. Biol., 245, 43-53, 1995; G. Jones, P.Willett, R. C. Glen, A. R. Leach and R. Taylor, J. Mol. Biol., 267,727-748, 1997; M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murrayand R. D. Taylor, Proteins, 52, 609-623, 2003). Either with or withoutmodifications of candidate or starting molecules, the modeled compoundis obtained.

It is understood that said molecular usually modeling makes use of theatomic coordinates of a 3-dimensional structure of said gp120 of HIV.Generally, the coordinates of a target molecule may be experimentallydetermined, e.g., by NMR spectroscopy and/or X-ray crystallography, ormay be obtained by molecular modeling, preferably homology modelingusing the high resolution structure of a first target molecule, saidhigh resolution structure being determined by experimental means, toestimate and calculate the structure of a second target molecule whichis different but related to the first target molecule and for which anexperimentally determined high resolution structure is not yetavailable. This way it is possible to study, e.g., the structure ofallelic variants of one protein without experimentally determining thestructure for every allelic variant but only one variant. Suitablesoftware is known in the art and includes, e.g., the GAMESS (US) QuantumChemistry package. The atomic coordinates of gp120 are accessible forthe person skilled in the art and can, e.g., be obtained from databasessuch as, e.g. the Brookhaven Protein Databank (PDB; www.pdb.org; e.g.,accession code 2B4C).

The term “inhibitor” refers to compounds lowering or abolishing theactivity of gp120, said activity being defined herein below in detail.Briefly, the activity of gp120 in the context of the present inventionmeans the capability of gp120 to bind to its receptor, i.e. theCD4-receptor or alpha4 beta7, on the surface of the target cell andthereby initiate viral entry. Methods to determine said activity ofgp120 are well-known in the art and described herein below. In preferredembodiments, inhibition effected by an inhibitor in accordance with theinvention refers to a reduction in activity of at least (for each value)10, 20, 30, preferred at least 40, 50, 60, 70, 80, 90, 95, 98 and morepreferred at least 99%. Most preferred, an inhibitor reduces theactivity to less than 10-2, less than 10-3, less than 10-4 or less than10-5 times as compared to the activity in the absence of the inhibitor.An inhibitor in accordance with the invention is capable of interactingwith a novel drug target site, i.e. said tunnel within gp120 that hasbeen surprisingly identified in the course of the invention.

The term “HIV (human immunodeficiency virus) glycoprotein (gp)120” isestablished in the art (cf. e.g., Coffin, Hughes, Varmus; Retroviruses;Cold Spring Harbor Laboratory Press; ISBN 0-87969-571-4) and has thesame meaning in the present application. gp120 is a glycoprotein exposedon the surface of the HIV envelope. The designation “120” stems from itsmolecular weight of 120 kilodaltons. gp120 is organized with an outerdomain, an inner domain with respect to its termini and a bridgingsheet. The gp120 gene is around 1.5 kb long and codes for around 500amino acids (cf. SEQ ID NOs: 2 for HIV-1). It is suggested that threecopies of gp120 form into a trimer that caps the end of gp41, anotherHIV surface glycoprotein that becomes exposed upon conformational changeof gp120 as a result of its interaction with the CD4-receptor (cf. ZhuP. et al., Plos Pathogens, 4(11):e1000203 (2008)). The above holds truefor HIV-1 and HIV-2 gp120 which represent the gp120 molecules of the twodifferent HIV species of HIV. Both species are well-known andcharacterized in the art. When referring in this specification only to“gp120” this is meant to refer to the gp120 molecule of both HIVspecies, i.e. HIV-1 gp120 and HIV-2 gp120; unless it is evident from thecontext that it is intended to refer to the gp120 of one given HIVspecies only.

The term “CD4-receptor” is also well-known in the art and has the samemeaning in accordance with the invention. Briefly, CD4 (cluster ofdifferentiation 4) is a glycoprotein expressed on the surface of Thelper cells, regulatory T cells, monocytes, macrophages and dendriticcells (cf., e.g., C. Janeway, Immunobiology, Garland Science; 6thedition; Part III, Chapter 6; ISBN-10: 0815341016; ISBN-13:987-0815341017). It is a co-receptor that assists the T cell receptor(TCR) in activating its T cell following an interaction with an antigenpresenting cell. CD4 can interact directly with MHC class II moleculeson the surface of the antigen presenting cell using its extracellulardomain. CD4 is a member of the immunoglobulin superfamily. It has fourimmunoglobulin domains (D1 to D4) that are exposed on the extracellularsurface of the CD4+ cell. D1 and D3 resemble immunoglobulin variable(IgV) domains, D2 and D4 resemble immunoglobulin constant (IgC) domains.As previously described and well-known in the art, CD4 is a primaryreceptor used by HIV to gain entry into host cells (cf., e.g., Bour S.et al., Microbiol Rev., 59(1):63-93 (1995)) working in concert withco-receptors as described above.

The term “integrin alpha4 beta7” is also termed integrin a4b7 (alsoknown as lymphocyte Peyer patch adhesion molecule (LPAM)) is well-knownin the art and has the same meaning in accordance with the invention.Integrin a4b7 is a heteromer receptor (being composed of a4 and b7receptor subunits) and is expressed on lymphoctyes and is thought to beresponsible for T-cell homing into gut-associated lymphoid tissues.

The term “interact” or “interaction” as used herein refers to a relationbetween at least two molecular entities. This relation may inter alia bedescribed in terms of intermolecular distances and/or free energies ofinteraction. In the first case, an interaction may be defined by atleast one intermolecular distance, preferably by more than one such astwo, three, four, five or more intermolecular distances. If aninteraction according to the invention is to be described in terms ofintermolecular distances only, it is envisaged to use at least threesuch distances. Typically, intermolecular distances are determined asdistances between the centers of atoms of the respective interactingmolecular entities. In this case, intermolecular distances according tothe invention are referred to as interatomic distances. Preferably, asuch determined interatomic distance is equal or less than 8, 7, 6, 5,or 4 Angströms, more preferably in the range from 3.6 and 2.8 Angströms.Preferred values include 3.4, 3.2 and 3.0 Angströms. Alternatively or inaddition, an interaction may be defined in terms of free energy. Thefree energy may be a total free energy determining the strength of anintermolecular interaction in its entirety or a partial free energy,said partial free energy resulting from, for example, one atom-atominteraction within a plurality of atom-atom interactions within theintermolecular interaction under consideration. Preferably, the totalfree energy of an interaction according to the invention is at least 100kJ/mol. More preferred are total free energies of an interaction of atleast 100, at least 150 or at least 200 kJ/mol.

As an alternative or additional parameter, the IC50 concentration may beused to characterize the strength of an intermolecular interactionbetween inhibitor and gp120. The IC50 concentration is the concentrationof an inhibitor that is required to inhibit 50% of the target'sactivity, in the present case the activity of gp120. Preferably, themodeled inhibitor interacts with said at least two motifs such that theIC50 concentration is in the two-digit micromolar range, i.e. below 100μM. More preferred are IC50 concentrations below 50 μM, below 10 μM orbelow 1 μM. Yet more preferred are nanomolar or even picomolarconcentrations, e.g. inhibitors with an IC50 concentration below 100 nM,below 10 nM, below 1 nM or below 100 pM. More generally speaking, andapplicable to any inhibitor referred to in this specification, theconcentration that is required to achieve an inhibition of 50% of thetarget's activity may be used. Preferred values of IC50 concentrationsas recited above apply also to these concentrations.

An intermolecular interaction may comprise one or more types ofinteractions. Types of interactions include charge-charge,charge-dipole, and dipole-dipole interactions and furthermore hydrogenbonds and hydrophobic interactions. Dipoles may be permanent, induced orfluctuating. Interactions involving permanent dipoles and hydrogen bondsmay be of particular relevance, since they are capable of specificallypositioning and orienting a ligand or modulator in a binding pocket ortunnel. Interactions such as, e.g., described above may also be groupedinto and referred to as covalent or non-covalent interactions or bonds.A “covalent” interaction is a form of chemical bonding that ischaracterized by the sharing of pairs of electrons between atoms, orbetween atoms and other covalent bonds. Covalent bonding includes manykinds of interaction well-known in the art such as, e.g., σ-bonding,π-bonding, metal to non-metal bonding, agostic interactions andthree-center two-electron bonds. A “non-covalent” bond is a chemicalbond that does not involve the sharing of pairs of electrons.Non-covalent bonds are critical in maintaining the three-dimensionalstructure of large molecules, such as proteins and nucleic acids, andare involved in many biological processes in which molecules bindspecifically but transiently to one another. There are several types ofnon-covalent bonds: hydrogen bonding, ionic interactions, Van-der-Waalsinteractions, charge-charge, charge-dipole, dipole-dipole bonds andhydrophobic bonds. Non-covalent interactions often involve severaldifferent types of non-covalent bonds working in concert, e.g., to keepa ligand in position on a target binding site. An interaction may occurwith a group such as a charge or a dipole, which may be present manytimes at the surface of the target molecule. Preferably, an interactionis specific, i.e., it occurs at a defined site of the target molecule,i.e. gp120, and goes along with the formation of a network of severaldistinct and specific interactions. While a specific interaction mayoccur with hardly any change of the conformation of the moleculesinvolved (“key-in-lock”), in accordance with the present invention itinvolves conformational changes of one or both of the binding partners(“hand-in-glove” paradigm). The term “binding”, for example as usedherein below with regard to the binding of CD4 or integrin a4b7 togp120, is meant to refer to such a specific interaction, i.e. “binding”is a specific form of interaction. Interaction of the modeled compoundwith the tunnel of gp120 in accordance with the invention may includeone or more types of interaction described above (provided that said oneor more types of interaction involve interatomic distances of less than8 Angström) and results in the positioning of the modeled compoundpartially or fully in the tunnel within gp120 so that it leads to theinhibition of the binding of gp120 to CD4 or integrin a4b7. Saidinhibition is due to the inability of gp120—once saidmodeled/synthesized compound is correspondingly positioned—to undergo aconformational change necessary for binding to CD4 or integrin a4b7.While not wishing to be bound to a specific theory, it is understood inaccordance with the present invention that a compound interacting withat least two amino acid residues comprised in the six motifs in a gp120molecule will be able to inhibit binding to CD4 and at the same time tointegrin a4b7. This is because the flexibility of the tunnel withingp120 is considered to be key with regard to the adoption of anyconformation of gp120. It is further understood that some combinationsof motifs and some combinations of interactions sites in said motifs mayprove more potent, i.e. inhibit binding to a higher degree, when ininteraction with a suitable modeled/synthesized compound with regard toa either CD4 or integrin a4b7, but are nevertheless expected to have aninhibitory effect on both.

The term “motif” as used in accordance with the present invention iswell-known in the art with regard to molecular modelling. For example,in Oliva et al., J Mol Biol 266(4): 814-830 (Mar. 7, 1997) the natureand structural make-up of motifs involved in forming protein loops isextensively discussed; the disclosure content of Oliva et al. isexpressly incorporated herein by reference. The term “comprises theamino acid sequence” in accordance with the invention is meant to referto those amino acids that make up the motif.

Interaction of the modelled compound according to the invention is tooccur with an at least two interaction sites comprised in the six motifsor variants thereof defining the 3-dimensional structure of a tunnelwithin gp120. This includes interaction with at least two interactionsites in one motif or in any combination of motifs, e.g., withinteraction sites in the first and second motif; with interaction sitesin the first and third motif; with interaction sites in the first andfourth motif; with interaction sites in the first and fifth motif; withinteraction sites in the first and sixth motif; with interaction sitesin the second and third motif; with interaction sites in the second andfourth motif; with interaction sites in the second and fifth motif; withinteraction sites in the second and sixth motif; with interaction sitesin the third and fourth motif; with interaction sites in the third andfifth motif; with interaction sites in the third and sixth motif; withinteraction sites in the fourth and fifth motif; with interaction sitesin the fourth and sixth motif; or with interaction sites in the fifthand sixth motif. Also envisaged are combinations of interactionsinvolving combinations of binding sites in three motifs, four, five orall six motifs. Preferred are combinations of motifs in which at leastthe second, third or fourth motif is present. For example, preferred isthe combination of the second and third motif; the second and fourthmotif; the third and fourth motif; or the second, third and fourthmotif. In accordance with the invention the modelled/synthesizedcompound preferably interacts with an interaction site in a motif,wherein said interaction site minimally comprises one amino acidresidue, but may also comprises at least (for each value) 30%, 40%, 50%,60%, 70%, 80% or at least 90% or all amino acid residues of a motif.Thus, referring to an interaction with at least two interaction sitesrefers to an interaction with at least two amino acid residues comprisedwithin the motifs making up the tunnel. If said at least two amino acidresidues that said compound is to interact with are positioned on onemotif, in particular if adjacent, they may, however, be considered torepresent one interaction site. Said amino acid residues making up aninteraction site—when less than 100%—may be a consecutive stretch ofamino acid residues or may be an arrangement of amino acid residuesdispersed throughout a respective motif or combinations thereof, i.e. aninteraction site comprises single amino acid residues and consecutiveamino acid residues of a respective motif. The number of amino acidresidues interacted with in an interaction site in a motif by themodelled inhibitor may vary for each motif and explicitly comprises anycombination of the aforementioned percent values, and may also comprise,e.g., one, at least (for each value) two, three, four, five, six, seven,eight, nine, ten, 11, 12 or 13 amino acid residues as a continuousstretch or separated by amino acid residues that are not interactedwith. Preferably, the interaction site of the first motif is located inthe amino acid residue stretch made up of or at least partially involvesamino acids IISLWDQSLKPCVKL of the first motif (residues 2-16 of SEQ IDNO: 3) or variants thereof; the interaction site for the second motif islocated in the amino acid residue stretch made up of or at leastpartially involves amino acids VVSTQ of the second motif (residues 14-18of SEQ ID NO: 4) or variants thereof; the interaction site for the thirdmotif is located in the amino acid residue stretch made up of or atleast partially involves amino acids DPEIVMHSFNCGGEFFYC of the thirdmotif (residues 4-21 of SEQ ID NO: 5) or variants thereof; theinteraction site for the fourth motif is located in the amino acidresidue stretch made up of or at least partially involves amino acidsCPKISFEP of the fourth motif (SEQ ID NO: 6) or variants thereof; theinteraction site for the fifth motif is located in the amino acidresidue stretch made up of or at least partially involves amino acidsGGGDMRDN of the fifth motif (residues 4-11 of SEQ ID NO: 7) or variantsthereof; or the interaction site for the sixth motif is located in theamino acid residue stretch made up of or at least partially involvesamino acids CSS of the sixth motif or variants thereof. Hence, it isunderstood in accordance with the present invention that an interactionsite in a motif may exclusively involve amino acids from said preferredamino acid stretch, a combination of amino acids from said preferredamino acid stretch and amino acids chosen from the remaining amino acidresidues making up a given motif or only said remaining amino acidresidues, i.e. those not defined as belonging to said preferred aminoacid stretch in a given motif. Also preferred is that the interactionsite comprises or consists of only those amino acid residues that havebeen shown to be most conserved for HIV-1 and HIV-2. Such as forexample, the underlined amino acid residues of the first motifDIISLWDQSLKPCVKLT (SEQ ID NO: 3) for a gp120 of HIV-1 andDIISLWDQSLKPCVKLT (SEQ ID NO: 9) for a gp120 of HIV-2; the underlinedamino acid residues of the second motif NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQID NO: 4) for a gp120 of HIV-1 and NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ IDNO: 10) for a gp120 of HIV-2; the underlined amino acid residues of thethird motif SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5) for a gp120 of HIV-1and SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 11) for a gp120 of HIV-2; theunderlined amino acid residues of the fourth motif CPKISFEP (SEQ ID NO:6) for a gp120 of HIV-1 and CPKISFEP (SEQ ID NO: 14) for a gp120 ofHIV-2; the underlined amino acid residues of the fifth motifFRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) for a gp120 of HIV-1 andRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 12) for a gp120 of HIV-2; theunderlined amino acid residues of the sixth motif CSS for a gp120 ofHIV-1 and CSS for a gp120 of HIV-2. The underlined amino acid residuesin motifs of gp120 of HIV-1 have been identified to be conservedthroughout various gp120 HIV-1 variants to a degree of at least 70%, atleast 80 or at least 90% (cf. FIGS. 15 to 19). The underlined amino acidresidues in motifs of a gp120 of HIV-2 have been identified to beconserved throughout various HIV-2 variants to a degree of at least 70%,at least 80 or at least 90% (cf. FIGS. 23 to 25). Accordingly, theunderlined amino acid residues show a high degree of cross-speciesconservation. More preferred is that an interaction site of a givenmotif consists of or comprises the above-defined amino acid residues ora preferred stretch in a motif that belong to the above-defined mostconserved amino acids within the respective motif. While not wishing tobe bound to a specific scientific theory, it is understood that aninhibitor that interacts with interaction sites involving partially orcompletely identical amino acid residues for each HIV species is aninhibitor that is expected to act as potent cross species inhibitor. Itis, however, equally understood that an inhibitor interacting with aninteraction site involving completely or partially different amino acidresidues for each HIV species is also expected to act as a potent crossspecies inhibitor based on the reasoning that it is likely that variantamino acids may have characteristics as the amino acid they replace suchas, e.g., their overall or partial electric charge, and hence cansubstitute each other. Since the replacing amino acids are also capableof forming a tunnel in concert with the other motifs, it can be expectedthat an inhibitor will not necessarily be affected by the exchange.

The amino acid sequence of the first to sixth motif forming the3-dimensional structure of a tunnel within gp120 of HIV-1 has beendetermined with regard to a specific sequence of a specific HIV-1 strain(SEQ ID NO: 2; Protein data bank accession number 2B4C_G). It could bedemonstrated by cross population analysis of about 80,000 amino acidsequences of gp120 of HIV-1 variants (taken from the National Institutesof Health databank) that various single or continuous stretches of aminoacids in the sequence of the motifs are to a high degree, i.e. at least90% (corresponding to a conservation index of 9 as will be explainedbelow) of variant motifs, carry the identical amino acid as in thesequence of the motifs mentioned herein above, conserved throughout theanalysed population (cf. FIGS. 15 to 17 and 19 and 23 to 24 A and 25).The sequence of some of the motifs identified for gp120 of HIV-1 weresubsequently compared to the about 500 available amino acid sequencesfor gp120 of HIV-2 resulting in the finding that i) also in gp120 ofHIV-2 motifs exist that define a similar tunnel as in gp120 of HIV-1 andii) some amino acids are conserved across the HIV-1 to HIV-2 speciesborder. On the basis of the population analysis, it can be expected thatmotifs in various HIV-1 strains will be composed of the same or verysimilar (variants thereof) amino acid sequences as defined for eachmotif herein above.

It is understood in accordance with the present invention and due to thewell-known variant nature of the gp120 molecule in the various strainsof the HIV species, that the above described amino acid sequences ofeach of the five motifs may vary to different extents, i.e. form“variants thereof” that have retained the capability of forming saidinteraction sites within the 3-dimensional structure of said tunnelwithin said gp120. Preferably, said variants are natural variants, i.e.motifs that are part of the gp120 sequence of naturally occurring HIVvariants. The sequence of said variants can be determined by aligningsequences, wherein methods for sequence alignment are well-known in theart and described herein below. First, the amino acid sequence of agp120 of a given strain is obtained, e.g., by protein sequencing (e.g.,by mass spectrometry or Edman degradation). Next, the identity of thesequence is compared by aligning the sequence to a gp120 amino acidsequence comprising the motifs as specified herein above, e.g. in SEQ IDNO:2. Then, the sequence of the variant strain is determined at aposition corresponding the position of the motif within the sequence ofSEQ ID NO:2. The determination of said corresponding position may beeffected by comparing the aligned sequence next to the motif in SEQ IDNO:2 for the presence of identical sequences. The position of the firstmotif with regard to SEQ ID NO: 2 is from the amino acid residue atposition 29 to 45; of the second motif with regard to SEQ ID NO: 2 isfrom the amino acid residue at position 99 to 125; of the third motifwith regard to SEQ ID NO: 2 is from the amino acid residue at position221 to 242; of the fourth motif with regard to SEQ ID NO: 2 is from theamino acid residue at position 63 to 70; of the fifth motif with regardto SEQ ID NO: 2 is from the amino acid residue at position 320 to 341;of the sixth motif with regard to SEQ ID NO: 2 is from the amino acidresidue at position 297 to 299. Having identified said correspondingposition, the identity with regard to amino acid residue and length ofthe motif can be determined. A sequence variant can be up to (for eachvalue) 3, 2 or up to 1 amino acid residue shorter or longer than thecorresponding motif. Hence, a motif variant may in addition to having asequence variation or alternatively thereto comprise up to (for eachvalue) 1%, 2%, 4%, 6%, 8%, 10%, 15% or up to 20% less amino acidresidues as compared to the motifs defined herein above or comprise upto (for each value) 1%, 2%, 4%, 6%, 8%, 10%, 15% or up to 20% more aminoacid residues as compared to the motifs defined herein above. Alsoenvisaged is that the variants of motifs as defined herein above maycomprise a sequence of a given motif that is only at least (for eachvalue) 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or 99° A identical compared to the sequence of the corresponding motifas defined herein above. While differing with regard to the identity ofamino acid residues, it is understood that a given variant motifnevertheless comprises amino acids that are related, preferably, interms of their structure and charge, to the amino acid residues theyreplace (in comparison to the herein above defined amino acids) andtherefore do not compromise the 3-dimensional integrity of the tunnelwithin a gp120 molecule. In other words, it must be capable of definingsaid interaction sites within the 3-dimensional structure of said tunnelwithin said gp120. Such amino acids may be, e.g., those capable ofestablishing non-covalent interactions with other parts of a given motifand/or the remaining gp120 molecule so as to maintain the 3-dimensionalstructure of at least the tunnel. The skilled person is well-aware ofamino acids that may be grouped according to their relevantcharacteristics, such as e.g., their overall or partial charge.Furthermore, the skilled person is in the position to identify suitableamino acid residues in silico that do not influence the 3-dimensionalintegrity of said tunnel within gp120 of HIV.

In FIGS. 15 to 17 and 19 (HIV-1) and 23 to 24A and 25 (HIV-2) thevariant amino acids for each position in a given motif are provided inorder of likelihood taking into account the sequences of the availableHIV-1 and HIV-2 gp120 sequences for each species. Variants of the firstmotif for gp120 of HIV-1 (SEQ ID NO: 15) can have the amino acid residueE, T, A, Q, N, V, K, S, G or L at the first position of said motif; theamino acid residue Y, V, L, H, S, T, N, K, A, M or D at the secondposition of said motif; the amino acid residue A, V, T, K, L, S, N, Q,R, Y or E at the third position of said motif; the amino acid residue L,P, T, A, Q, N, F, I, E, G, R, K or D at the fourth position of saidmotif; the amino acid residue F, I, V, M, S, G, R, T, C, P, K, Y or D atthe fifth position of said motif; the amino acid residue Y, D, T, K, E,N, R, Q, G, H, S, M, F or L at the sixth position of said motif; theamino acid residue K, N, E, S, R, G, T, A, Q, F or Y at the seventhposition of said motif; the amino acid residue L, E, N, K, S, T, D, A,R, G, P, H, Y, I, M or F at the eighth position of said motif; the aminoacid residue D, E, K, N, G, T, A, M, L, R, Q, F, V or H at the ninthposition of said motif; the amino acid residue V, S, N, I, M, T, D, H,Q, Y, P, R, E, G, A, F or K at the 10th position of said motif; theamino acid residue V, E, S, T, N, Q, A, I, R, F, G, Y, M, L, P or D atthe 11th position of said motif; the amino acid residue S, N, T, E, G,Y, K, R, Q, D, L, A or V at the 12th position of said motif; the aminoacid residue I, T, E, M, S, L, Y, A, K, N, R, F, D, G, H, V, W or Q atthe 13th position of said motif; the amino acid residue Y, D, I, L, S,T, E, A, G, N, R, K, H, C, M, F or Q at the 14th position of said motif;the amino acid residue R, N, E, T, D, Q, G, L, S, I, P, Y, A or V at the15th position of said motif; the amino acid residue N, T, D, I, E, S, A,D, Y, K, G, V, R, M, F, Q or H at the 16th position of said motif;and/or the amino acid residue I, D, V, N, S, R, Y, K, E, M, H, G, A, Lor C at the 17th position of said motif, wherein D of the motif takesthe first position and T takes the 17th position of the first motif.

Variants of the second motif for gp120 of HIV-1 (SEQ ID NO: 16) can havethe amino acid residue K, D, S, P, E, R, T, Q, H, G, I, M, F, Y or V atthe first position of said motif; the amino acid residue I, A, G, S, F,T, L, C, K, Y, Q or E at the second position of said motif; the aminoacid residue T, G, N, R, C, A, V, I, L, F or K at the third position ofsaid motif; the amino acid residue S, V, I, A, P, H, Q, L, G, M, K or Rat the fourth position of said motif; the amino acid residue I, A, L, G,Y, E, R, S, T or F at the fifth position of said motif; the amino acidresidue T, L, H, R, P, A, V, I, K, S, N or E at the sixth position ofsaid motif; the amino acid residue R, Y, W, G, F, S, M, A, V, I, P or Kat the seventh position of said motif; the amino acid residue A, P, S,Q, I, R, Y, V, G or H at the eighth position of said motif; the aminoacid residue Q, R, Y, P, N, L, D, T, G, M, V, W, S or E at the ninthposition of said motif; the amino acid residue R, E, S, W, A, V, P or Nat the 10th position of said motif; the amino acid residue V, T, L, N,F, M, S, G, A, R or D at the 11th position of said motif; the amino acidresidue K, M, S, Q, T, N, G, E, L, A, W, I, P or H at the 12th positionof said motif; the amino acid residue A, S, L, Q, T, R, H or E at the13th position of said motif; the amino acid residue T, A, I, M, L, G, E,S, P, Q, C, W, H or R at the 14th position of said motif; the amino acidresidue I, A, L, M, G, T, E, S, P, F, C, W, R, Y or N at the 15thposition of said motif; the amino acid residue T, P, L, A, V, Q, I, F,N, H, K or Y at the 16th position of said motif; the amino acid residueS, A, I, N, P, L, H, G, V, R or Q at the 17th position of said motif;the amino acid residue H, R, S, P, K, A, E, L, T, N or M at the 18thposition of said motif; the amino acid residue F, P, V, M, S, I, T, A,C, Q, R, Y, W or H at the 19th position of said motif; the amino acidresidue I, P, V, A, T, M, S, Q; R, C, F, Y, G, N, W or K at the 20thposition of said motif; the amino acid residue V, F, I, S, M, P, W, Y,G, T, Q, C or E at the 21st position of said motif; the amino acidresidue D, S, K, P, Y, H, T, I, M, F, A, G, L, W or E at the 22ndposition of said motif; the amino acid residue P, S, A, D, C, V, W, R,N, F or E at the 23rd position of said motif; the amino acid residue T,G, R, N, C, I, A, V, Q, L, P, K, M or D at the 24th position of saidmotif; the amino acid residue I, V, T, R, P, Q, S, F, A, Y, M, G, K or Eat the 25th position of said motif; the amino acid residue S, T, V, P,E, G, D, Q, L, R, K, Y, N, C, M or F at the 26th position of said motif;and/or the amino acid residue K, G, A, R, D, T, Q, V, N, S, L, I, H or Yat the 27th position of said motif, wherein N of the motif takes thefirst position and E takes the 27th position of the second motif.

Variants of the third motif for gp120 of HIV-1 (SEQ ID NO: 17) can havethe amino acid residue A, T, P, V, L, I, Q, R, K, N, G, E, H, Y, F, M,W, D or C at the first position of said motif; the amino acid residue E,R, D, S, P, A, K, L, Q, N, V, W, I, H or T at the second position ofsaid motif; the amino acid residue E, R, K, S, W, V, L, A, N, T, I, F, Dor Q at the third position of said motif; the amino acid residue G, T,N, A, E, H, V, I, S, R, Y, P, F, Q, L, K or M at the fourth position ofsaid motif; the amino acid residue L, I, V, S, Q, A, M, T, R; E; D, F,H, Y, K, W, N, C or G at the fifth position of said motif; the aminoacid residue K, Q, G, D, R, V, N, A, P, S, L, I, H, T or Y at the sixthposition of said motif; the amino acid residue V, L, T, F, N, M, S, A,Y, G, P, K, D, C, H or R at the seventh position of said motif; theamino acid residue T, I, E, A, M, K, S, L, R, Q, P, Y, C, G, N, H, F orD at the eighth position of said motif; the amino acid residue T, L, R,K, S, A, H, Q, V, I, F, N, P, Y, E, C, G, D or W at the ninth positionof said motif; the amino acid residue L, Y, F, R, I, P, T, S, N, Q, V,D, A, G, M, C, W or E at the 10th position of said motif; the amino acidresidue T, H, N, I, M, R, G, F, Y, L, A, Q, K, C, V, P, D, W or E at the11th position of said motif; the amino acid residue V, L, S, I, C, Y, H,T, R, W, G or N at the 12th position of said motif; the amino acidresidue T, I, S, C, D, Y, V, H, K, F, L, M, P, R, G; Q; E or A at the13th position of said motif; the amino acid residue R, W, Y, S, V, G, L,F, I, H, A, N or Q at the 14th position of said motif; the amino acidresidue R, A; K, Q, H, E, M, N, V, S, T, W, Y, C, I, D, L or F at the15th position of said motif; the amino acid residue E, R, K, W; V, S, A,L, N, D, I, F or Y at the 16th position of said motif; the amino acidresidue G, K, D, N, R, V, A, L, F, S, Q, W, H, Y, C, M or T at the 17thposition of said motif; the amino acid residue L, S, I, Y, V, C, N, G,M, W or Q at the 18th position of said motif; the amino acid residue L,S, Y, V, P, I, C, D, A, M or N at the 19th position of said motif; theamino acid residue F, N, C, H, S, L, V; I, T, P, G, M, W, K, D or E atthe 20th position of said motif; the amino acid residue R, Y, L, S, W,G, A, F, V, I, M, T, P or K at the 21st position of said motif; and/orthe amino acid residue D, S, K, Y, T, E, I, H, G, C, Q, R, A; V, P, M orF at the 22nd position of said motif, wherein S of the motif takes thefirst position and N takes the 22nd position of the third motif.

Variants of the fifth motif for gp120 of HIV-1 (SEQ ID NO: 18) can havethe amino acid residue L, V, I, S, T, Y, C, M, A, P, N, W, H, R or D atthe first position of said motif; the amino acid residue G, K, T, S, I,Y, Q, L, E, P, V, C, W, D, M, F or N at the second position of saidmotif; the amino acid residue L, S, A; D, R, H, T, Q, N, G or E at thethird position of said motif; the amino acid residue A, E, I, V, T, Q,L, R, S, W, M, P, K or N at the fourth position of said motif; the aminoacid residue R, E, S, A, L, K, C, P, N or Q at the fifth position ofsaid motif; the amino acid residue E, R, K, S, A, D, C or N at the sixthposition of said motif; the amino acid residue N, E, G, Y, S, I, H, R,A, V, Q, T or K at the seventh position of said motif; the amino acidresidue I, T, V, L, R, Y, K, F, G or S at the eighth position of saidmotif; the amino acid residue K, M, Q, G, E, V, T, N, W, I, F or S atthe ninth position of said motif; the amino acid residue N, E, G, V, H,A, L, C, W, R, K, S, T or Y at the 10th position of said motif; theamino acid residue I, S, D; K, L, H, Y, T, Q, G, R, A; C or M at the11th position of said motif; the amino acid residue S, R, G; N, C, L, F,P or K at the 12th position of said motif; the amino acid residue K, G,A, S, E, T, L or I at the 13th position of said motif; the amino acidresidue N, T, G, R, Q, M, D, I, C, K, Y, A, V or H at the 14th positionof said motif; the amino acid residue K, Q, G, D, R, A; V, N, H or M atthe 15th position of said motif; the amino acid residue I, F, S, V, M,P, K, N, Q or E at the 16th position of said motif; the amino acidresidue F, L, H, C, S, D, I, A, V, N or E at the 17th position of saidmotif; the amino acid residue R, N, Q, E, T, Y, P, G, V, L, I, M, S or Dat the 18th position of said motif; the amino acid residue H, C, N, F,S; I or E at the 19th position of said motif; the amino acid residue R,E, N, T, Q, G, I or P at the 20th position of said motif; the amino acidresidue I, T, A, L, G, M, S or D at the 21st position of said motif;and/or the amino acid residue I, A, L, E, G, S, M or K at the 22ndposition of said motif, wherein F of the motif takes the first positionand V takes the 22nd position of the fifth motif.

Variants of the first motif for gp120 of HIV-2 (SEQ ID NO: 19) can havethe amino acid residue R, S or T at the first position of said motif;the amino acid residue Y, V or W at the second position of said motif;the amino acid residue N, W or Y at the third position of said motif;the amino acid residue E or N at the fourth position of said motif; theamino acid residue T or K at the fifth position of said motif; the aminoacid residue F at the sixth position of said motif; the amino acidresidue Y or E at the seventh position of said motif; the amino acidresidue S, T or V at the eighth position of said motif; the amino acidresidue V or T at the ninth position of said motif; the amino acidresidue D, I or C at the 10th position of said motif; the amino acidresidue V or E at the 11th position of said motif; the amino acidresidue V or T at the 12th position of said motif; the amino acidresidue T at the 13th position of said motif; the amino acid residue D,E or R at the 14th position of said motif; the amino acid residue N or Eat the 15th position of said motif; the amino acid residue N or S at the16th position of said motif; and/or the amino acid residue S or Q at the17th position of said motif, wherein D of the motif takes the firstposition and T takes the 17th position of the first motif.

Variants of the second motif for gp120 of HIV-2 (SEQ ID NO: 20) can havethe amino acid residue K, R or S at the first position of said motif;the amino acid residue I or K at the second position of said motif; theamino acid residue V, I or F at the third position of said motif; theamino acid residue A, V or S at the fourth position of said motif; theamino acid residue A, S, T or G at the fifth position of said motif; theamino acid residue T, S or A at the sixth position of said motif; theamino acid residue R or L at the seventh position of said motif; theamino acid residue A or Y at the eighth position of said motif; theamino acid residue R or G at the ninth position of said motif; the aminoacid residue M, V, E, L, I or Q at the 10th position of said motif; theamino acid residue M, R; K or T at the 11th position of said motif; theamino acid residue E, K or G at the 12th position of said motif; theamino acid residue T, S, A or M at the 13th position of said motif; theamino acid residue Q at the 14th position of said motif; the amino acidresidue T, S, A, P at the 15th position of said motif; the amino acidresidue at the 16th position of said motif is not variant; the aminoacid residue A, M or S at the 17th position of said motif; the aminoacid residue W at the 18th position of said motif; the amino acidresidue F or S at the 19th position of said motif; the amino acidresidue G or A at the 20th position of said motif; the amino acidresidue F at the 21st position of said motif; the amino acid residue Cor S at the 22nd position of said motif; the amino acid residue S or Pat the 23rd position of said motif; the amino acid residue T, A or I atthe 24th position of said motif; the amino acid residue R or K at the25th position of said motif; the amino acid residue S at the 26thposition of said motif; and/or the amino acid residue G, K, V or D atthe 27th position of said motif, wherein N of the motif takes the firstposition and E takes the 27th position of the second motif.

Variants of the third motif for gp120 of HIV-2 (SEQ ID NO: 21) can havethe amino acid residue A, K, R, E, G, T, Q, V, D, P or N at the firstposition of said motif; the amino acid residue P, S, D; N or K at thesecond position of said motif; the amino acid residue S, A, P or R atthe third position of said motif; the amino acid residue N, G, or S atthe fourth position of said motif; the amino acid residue A, G or S atthe fifth position of said motif; the amino acid residue D, P, K or Q atthe sixth position of said motif; the amino acid residue V, A, T or E atthe seventh position of said motif; the amino acid residue A, T, R, E,M, K or S at the eighth position of said motif; the amino acid residueY, F or H at the ninth position of said motif; the amino acid residue M,L or T at the 10th position of said motif; the amino acid residue W or Rat the 11th position of said motif; the amino acid residue T, S or I atthe 12th position of said motif; the amino acid residue D at the 13thposition of said motif; the amino acid residue at the 14th position ofsaid motif is not variant; the amino acid residue R, M, K or Q at the15th position of said motif; the amino acid residue R at the 16thposition of said motif; the amino acid residue K or D at the 17thposition of said motif; the amino acid residue L or S at the 18thposition of said motif; the amino acid residue L, P, S or Y at the 19thposition of said motif; the amino acid residue H or C at the 20thposition of said motif; the amino acid residue at the 21st position ofsaid motif is not variant; and/or the amino acid residue D, S or K atthe 22nd position of said motif, wherein S of the motif takes the firstposition and N takes the 22nd position of the third motif.

Variants of the fifth motif for gp120 of HIV-2 (SEQ ID NO: 22) can havethe amino acid residue N, S, I, Y, V, R or D at the first position ofsaid motif; the amino acid residue I, S, M or T at the second positionof said motif; the amino acid residue T, A, L or S at the third positionof said motif; the amino acid residue F, M, V, E, P or S at the fourthposition of said motif; the amino acid residue S, V, A, L, I or N at thefifth position of said motif; the amino acid residue A at the sixthposition of said motif; the amino acid residue E at the seventh positionof said motif; the amino acid residue V, L, Y or E at the eighthposition of said motif; the amino acid residue A, S, G or K at the ninthposition of said motif; the amino acid residue E, K or N at the 10thposition of said motif; the amino acid residue L, M or K at the 11thposition of said motif; the amino acid residue Y, F or N at the 12thposition of said motif; the amino acid residue K at the 13th position ofsaid motif; the amino acid residue L or V at the 14th position of saidmotif; the amino acid residue K at the 15th position of said motif; theamino acid residue P or S at the 16th position of said motif; the aminoacid residue G at the 17th position of said motif; the amino acidresidue D at the 18th position of said motif; the amino acid residue atthe 19th position of said motif is not variant; the amino acid residue Tor N at the 20th position of said motif; the amino acid residue L or Sat the 21st position of said motif; and/or the amino acid residue I atthe 22nd position of said motif, wherein F of the motif takes the firstposition and V takes the 22nd position of the fifth motif.

The amino acid residues are arranged in the order of likelihood. Inother words, the first mentioned amino acid residue for a given positionwithin a given motif is the amino acid residue most likely to replacethe amino acid at the corresponding position of said given motif in avariant motif based on a population analysis as described herein above.While the above covers sequence variants, the additional information ofthe likelihood of a given amino acid variation occurring will beappreciated by the person skilled in the art since it will be possibleto thereby design inhibitors which will interact with the mostfrequently occurring and/or rarely occurring HIV strains. Any of theabove described sequence variants is understood to be capable of forminga tunnel within said 3-dimensional structure of said gp120. The sequenceof said sequence variants is depicted in FIGS. 15 to 17 and 19 for HIV-1and in FIGS. 23 to 24A and 25 for HIV-2. In case of a discrepancy of theamino acid sequence information for the variants given in thedescription herein above and the sequence listing in comparison to saidFigures, the authoritative sequence is that given in said Figures.

The term “binding” as used in accordance with the present invention withrespect to the association of CD4 or integrin a4b7 and gp120 refers tothe specific interaction of CD4 or integrin a4b7 to gp120 when broughtinto contact. Said specific interaction is characterized by asubstantial conformational change of the core and periphery of gp120accompanying said interaction. Furthermore, due to the structuraladaptation of gp120 the kinetics of the interaction are slow (cf. MyszkaD. et al., Proc Natl Acad Sci U.S.A., 97(16):9026-9031 (2000); regardingCD4).

The term “peptidomimetic” as used in accordance with the inventionrefers to a compound that mimics the biological action of a natural(poly)peptide, i.e. gp120. Said compound may be a protein-like chaincontaining non-peptidic elements designed to mimic a (poly)peptide.Accordingly, a peptidomimetic in accordance with the present inventionmay be a compound that mimics the three-dimensional structure of a gp120as defined herein, and also mimicks its function. Specificallyencompassed are peptidomimetics that at least structurally andfunctionally mimic the part of gp120 that makes up the novel tunnelbinding site as described herein. The term “peptide” as used hereindescribes a group of molecules consisting of up to 30 amino acids,whereas “proteins” consist of more than 30 amino acids. Peptides andproteins may further form dimers, trimers and higher oligomers, i.e.consisting of more than one molecule which may be identical ornon-identical. The corresponding higher order structures are,consequently, termed homo- or heterodimers, homo- or heterotrimers etc.The terms “peptide” and “polypeptide” (wherein “polypeptide” isinterchangeably used with “protein”) also refer to naturally modifiedpeptides/proteins wherein the modification is effected e.g. byglycosylation, acetylation, phosphorylation and the like. Suchmodifications are well-known in the art.

The amino acid residues of the motifs of the main embodiment provide astructural description of a novel drug target site of gp120. Said noveldrug target site takes the form of a tunnel that is formed by said sixsequence motifs within gp120 that was identified by the inventors. Inother words, a “tunnel” in accordance with the invention consists ofsaid six motifs or variants thereof. Preferably, the motifs have asequence that has the same length as the specific sequence of the motifsrecited herein above. It is understood that the presence of all of themotifs or variants thereof is sufficient to form said tunnel within the3-dimensional structure of gp120. In other words, the tunnel is aninherent feature of the HIV strains analyzed.

Since at least two amino acid residues comprised in six motifs formingthe 3-dimensional structure of the tunnel interact with the compound tobe modeled, the present invention also provides an implicit definitionof pharmacophores capable of binding to said tunnel. The term“pharmacophore” is known in the art and refers to the molecularframework responsible for the biological or pharmacological activity ofa compound (Güner (2000), Pharmacophore Perception, Development, and usein Drug Design, ISBN 0-9636817-6-1; Langer and Hoffmann (2006),Pharmacophores and Pharmacophore Searches, ISBN 3-527-31250-1). Typicalpharmacophore features include hydrogen bond donors, hydrogen bondacceptors, dipoles, charges, ions and hydrophobic moieties. Thepharmacophore furthermore includes information on the spatialarrangement of one or more of such moieties.

The amino acid sequence of the motifs or variants thereof of the mainembodiment constitute the structural elements that form thethree-dimensional structure of a tunnel within gp120 which hassurprisingly not been identified prior to this invention despiteextensive research in the HIV research field. As shown in the enclosedfigures, these structural elements form a previously unknown interactionarea, i.e. said tunnel, of gp120. More surprisingly, this tunnel wasfound by the present inventors to be an allosteric interaction area. Theterm “allosteric” is known in the art and refers to an alteration ofconformation in response to ligand interaction. Ligand interactionoccurs at a site which is not the active site or one of the active sitesof the target (here gp120), but exerts a modulating effect on the activesite(s). This modulation involves structural changes which in turnfrequently entail functional changes. In other words, interaction ofligands with this tunnel reduces the ability of gp120 to undergoconformational changes required for its physiological action: it locks aconformation of gp120 that cannot mediate viral entry. The lockedconformation is an inactive conformation of gp120.

Sequence identity levels as recited above may be determined by methodswell known in the art. Two nucleotide or protein sequences can bealigned electronically using suitable computer programs known in theart. Such programs comprise BLAST (Altschul et al. (1990), J. Mol. Biol.215, 403-410), variants thereof such as WU-BLAST (Altschul & Gish(1996), Methods Enzymol. 266, 460-480), FASTA (Pearson & Lipman (1988),Proc. Natl. Acad. Sci. USA 85, 2444-2448), CLUSTALW (Higgins et al.(1994), Nucleic Acids Res. 22, 4673-4680) or implementations of theSmith-Waterman algorithm (SSEARCH, Smith & Waterman (1981), J. Mol.Biol. 147, 195-197). These programs, in addition to providing a pairwisesequence alignment (multiple sequence alignment in case of CLUSTALW),also report the sequence identity level (usually in percent identity)and the probability for the occurrence of the alignment by chance(P-value). Preferably, the BLAST program is employed to determinesequence identity levels.

Preferably the sequence identity at the amino acid sequence level ofgp120 is at least (for each value) 85%, 90%, more preferred at least95%, 96%, 97%, 98% and most preferred at least 99%. Preferred sequenceidentities at the nucleic acid sequence level are at least (for eachvalue) 85%, 90%, 92%, 94%, more preferred at least 95%, 96%, 97%, 98%and most preferred at least 99%. Nevertheless, sequence identities (forprotein and DNA sequences) of less than 80% such as e.g., at least (foreach value) 75%, 70%, 65%, 60%, 55%, 50%, 45%, or at least 40% areenvisaged. It is understood in accordance with the invention that thevariations in the amino acid sequence of said five sequence motifs, i.e.the amino acid sequence of the variants, is encompassed by saidaforementioned sequence identity levels.

The above definitions apply mutatis mutandis to other embodimentsdescribed herein below unless it is expressly stated otherwise.

The inventors have been able to identify a novel target site on gp120that provides the means to inhibit the binding of gp120 to CD4 orintegrin a4b7 resulting in the prevention of viral entry. The finding isparticularly surprising since gp120 has been the subject of intensestudies by research groups throughout the world for many years and isgenerally considered to be a structurally well defined molecule. In thisregard, many crystallization structures have been generated of gp120over the years and used in various studies to identify potential drugtargets on gp120. However, none of the prior art studies was able toidentify the tunnel as defined herein above constituting a novel drugtarget site on gp120.

Some prior art compounds have been shown to prevent the binding of HIV-1envelope gp120 protein to cellular CD4 receptors via a specific andcompetitive mechanism by binding to a binding site on the surface ofgp120 termed herein binding site I (Guo Q. et al., Journal of Virology,77(19):10582-10536 (2003)). Another group of inhibitors was found toprevent certain minimal conformational changes in gp120 upon gp120/CD4binding (Si Z. et al. Proceedings of the National Academy of Sciences,101(14):5036 (2004)). It was suggested that in this case an inhibitorbinds to another binding site on the surface of gp120 termed hereinbinding site II. The surface areas of the above-mentioned different twobinding sites have been shown to partially overlap in the region of asmall hydrophobic pocket (cf. FIG. 1). Although said binding sites havebeen studied extensively, the novel tunnel was not identified in any ofthe prior art studies regarding the structure of gp120 and compoundsbinding thereto. Without whishing to be bound by any specific theory,one reason for this may be that this tunnel opens and closesthermodynamically and therefore may only be visualized using, e.g.,computerised models which allow the protein to thermodynamically flex.The residues of binding site I are located in a large cavity which ispenetrated by the phenyl residue of CD4 and hence is responsible forgp120-CD4 interaction (cf. FIG. 2). A three-dimensional structure ofgp120 interacting with CD4 is presented in FIG. 3 (1 G9N from theProtein Data Bank (Wang R. et al., J. Med. Chem.,48(12):4111-4119(2005); Westbrook J. et al., Nucleic Acids Res.,31(1):489-491 (2003)). FIG. 3 shows the Phe43 residue of CD4 enteringdeep into a hydrophobic pocket within the gp120 structure around bindingsite I. Said pocket is naturally responsible for specific recognition ofthe Phe43 residue of CD4.

While employing molecular dynamics simulations to study various gp120conformations the inventors identified a water filled tunnel connectingthe hydrophobic pocket of binding site I with the hydrophobic pocket ofbinding site II. The tunnel has not been identified previously and ishence not present in any of the X-ray structures available in theProtein Data Bank. FIGS. 4 and 5 show that the binding sites I and IIare closely placed within the gp120 three-dimensional structure.

When targeting the tunnel with compounds that interact with, e.g. enter,the tunnel the inventors could show that said compounds reduce theflexibility of gp120 to the extent that a conformational changenecessary for binding to CD4 and to integrin a4b7 does not occur. As aresult the initiation of viral entry into the target cells is inhibited(see Examples 2 and 3).

Interestingly and supporting the functional principle of the presentinvention, the tunnel structure is to a significant degree conservedwithin the various HIV species and subspecies. This is evidenced by thestructural similarity of the HIV-1 and HIV-2 gp120 molecules which eachfeature the same tunnel elements, i.e. the motifs belonging to thetunnel as shown in the figures below (see also FIG. 22). While the aminoacids may vary to some extent from species to species or subspecies tosubspecies, the overall structure, i.e. presence of six motifs definingthe 3-dimensional structure of gp120, within one species including anysubspecies could be shown to be highly conserved (see FIG. 22). Withoutwishing to be bound by any specific scientific theory, the inventorsbelieve that the reason for the highly conserved structure within thegp120 molecule, viz. the tunnel, of various HIV strains and subtypes isthat it reflects an essential element of a universal, i.e. shared by allHIV strains and subtypes, mode of infection. In other words, disruptionof said tunnel by mutation appears to have inescapably led to anevolutionary dead end. Taking advantage of said evolutionary conservedfeature, the disruption of the functionality of the tunnel within gp120results in the prevention of interaction with CD4 and integrin a4b7.

As will immediately be appreciated by the person skilled in the art, theidentification of the above-described novel drug target on gp120 enablesthe development of novel compounds and their use as drugs in thetreatment of HIV infections and HIV-associated diseases as will bedescribed below. Corresponding compounds could overcome the problem ofdevelopment of resistant strains that is due to the knownhypervariability of existing drug target sites and would further affectgp120 without mechanism-related impacts on the immune system which areimplicit in the treatments targeting, e.g., CXCR4 and CCR5 co-receptors.In this regard, the inventors have conducted tests of antiviral activityof compounds interacting with the tunnel as described herein ondifferent HIV strains (see example 4) that demonstrated that the testedcompounds exhibit antiviral activity in different viral strains. Thiscorroborates the above hypothesis that the tunnel structure is a highlyconserved feature and as such a valuable cross-species and -strainpharmaceutical target.

In a preferred embodiment of the invention, the method further comprisessynthetically producing said designed inhibitor.

Accordingly, the invention also relates to a method of producing aninhibitor of the binding of HIV (human immunodeficiency virus)glycoprotein (gp)120 to a CD4-receptor or to the integrin alpha4 beta?(a4b7), the method comprising (a) the molecular modelling of a compoundsuch that the modelled compound interacts in silico with at least twoamino acid residues comprised in six motifs within the 3-dimensionalstructure of said gp120 of HIV or within a peptidomimetic reflecting the3-dimensional structure of said gp120 of HIV, wherein said interactionbetween said at least two amino acid residues and said compound ischaracterized by an interatomic distance of less than 8 Angströms,wherein a first motif of said six motifs comprises the amino acidsequence DIISLWDQSLKPCVKLT (SEQ ID NO: 3) or a variant thereof; whereina second motif of said six motifs comprises the amino acid sequenceNVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ ID NO: 4) or a variant thereof; whereina third motif of said six motifs comprises the amino acid sequenceSGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5) or a variant thereof; wherein afourth motif of said six motifs comprises the amino acid sequenceCPKISFEP (SEQ ID NO: 6) or a variant thereof; wherein a fifth motif ofsaid six motifs comprises the amino acid sequence FRPGGGDMRDNWRSELYKYKVV(SEQ ID NO: 7) or a variant thereof; and wherein a sixth motif of saidsix motifs comprises the amino acid sequence CSS or a variant thereof,said gp120 of HIV comprising or consisting of (i) the sequence of SEQ IDNO: 2; (ii) the sequence encoded by the sequence of SEQ ID NO: 1; (iii)a sequence being at least 50% identical to the sequence of SEQ ID NO: 2or to the sequence encoded by the sequence of SEQ ID NO: 1; or (iv) asequence encoded by a sequence being at least 50% identical to thesequence of SEQ ID NO: 1, wherein said sequence of (i), (ii), (iii) or(iv) comprises or encodes said motifs or variants thereof; and (b)synthesizing said compound that interacts with said at least two aminoacid residues comprise in said six motifs within the 3-dimensionalstructure of gp120.

These embodiments are particularly, but not exclusively, envisaged forthose cases where the inhibitor is a small organic molecule, a peptideor polypeptide. Means and methods for synthesizing peptides orpolypeptides are well known in the art and may involve organic synthesisand/or the recombinant production using the methods of molecular biologyand protein biochemistry. A large number of suitable methods exist inthe art to produce peptides or polypeptides in appropriate hosts. If thehost is a unicellular organism such as a prokaryote, a mammalian orinsect cell, the person skilled in the art can revert to a variety ofculture conditions. Conveniently, the produced protein is harvested fromthe culture medium, lysates of the cultured organisms or from isolated(biological) membranes by established techniques. In the case of amulticellular organism, the host may be a cell which is part of orderived from a part of the organism, for example said host cell may bethe harvestable part of a plant. A preferred method involves therecombinant production of protein in hosts as indicated above. Forexample, nucleic acid sequences comprising a (poly)nucleotide can besynthesized by PCR, and inserted into an expression vector. Subsequentlya suitable host may be transformed with the expression vector.Thereafter, the host is cultured to produce the desired peptide(s) orpolypeptide(s), which is/are isolated and purified.

An alternative method for producing peptides or polypeptides is in vitrotranslation of mRNA. Suitable cell-free expression systems for use inaccordance with the present invention include rabbit reticulocytelysate, wheat germ extract, canine pancreatic microsomal membranes, E.coli S30 extract, and coupled transcription/translation systems such asthe TNT-system (Promega).

These systems allow the expression of recombinant peptides orpolypeptides upon the addition of cloning vectors, DNA fragments, or RNAsequences containing coding regions and appropriate promoter elements.

In addition to recombinant production, the peptide or polypeptide may beproduced synthetically, e.g. by direct peptide synthesis usingsolid-phase techniques (cf Stewart et al. (1969) Solid Phase PeptideSynthesis; Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85(1963), 2149-2154).

Synthetic peptide or polypeptide synthesis may be performed using manualtechniques or by automation. Automated synthesis may be achieved, forexample, using the Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City Calif.) in accordance with the instructions providedby the manufacturer. Various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule. As indicated above, chemical synthesis, such as thesolid phase procedure described by Houghton Proc. Natl. Acad. Sci. USA(82) (1985), 5131-5135, can be used. Furthermore, (poly)peptides may beproduced semi-synthetically, for example by a combination of recombinantand synthetic production.

Peptide or polypeptide isolation and purification can be achieved by anyone of several known techniques; for example and without limitation, ionexchange chromatography, gel filtration chromatography and affinitychromatography, high pressure liquid chromatography (HPLC), reversedphase HPLC, and preparative disc gel electrophoresis. (Poly)peptideisolation/purification techniques may require modification of theproteins of the present invention using conventional methods. Forexample, a histidine tag can be added to the protein to allowpurification on a nickel column. Other modifications may cause higher orlower activity, permit higher levels of protein production, or simplifypurification of the protein.

As regards small organic molecules, reference is made to the Beilsteindatabase available from MDL Information Systems as an example.

In another preferred embodiment of the method of the invention, saidmolecular modelling comprises (a) measuring at least one intermoleculardistance; (b) calculating at least one free energy of interaction;and/or determining the accessibility to the tunnel.

Accessibility of the tunnel limits the size of the allosteric effector,i.e. the inhibitor, and thereby the maximal intermolecular interactionenergy. Tools for molecular modeling as described above typicallyprovide the option of measuring one or more intermolecular distances.Preferably, the intermolecular distances are determined as distancesbetween the centers of atoms. Tools for molecular modeling alsogenerally provide the option of calculating free energies ofinteraction. The term “free energy” in relation to an interaction iswell known in the art and is related to the equilibrium binding constantby the equation ΔG=−RT in K, wherein ΔG is the change in free energyupon binding, K is the binding constant, T is the temperature and R isthe universal gas constant. Free energies to be calculated may be, asdescribed above, total free energies and/or partial free energies.

Another embodiment of the invention relates to a method of identifyingan inhibitor of the binding of HIV (human immunodeficiency virus)glycoprotein (gp)120 to a CD4-receptor or to the integrin alpha4 beta?(a4b7), the method comprising (a) bringing into contact a HIV gp120 or apeptidomimetic reflecting the three-dimensional structure of said gp120and a test compound; (b) determining whether said test compoundinteracts with at least two amino acid residues comprised in six motifswithin the 3-dimensional structure of said gp120 or within apeptidomimetic reflecting the 3-dimensional structure of said gp120,wherein said interaction between said at least two amino acid residuesand said compound is characterized by an interatomic distance of lessthan 8 Angströms, wherein a first motif of said six motifs comprises theamino acid sequence DIISLWDQSLKPCVKLT (SEQ ID NO: 3) or a variantthereof; wherein a second motif of said six motifs comprises the aminoacid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ ID NO: 4) or a variantthereof; wherein a third motif of said six motifs comprises the aminoacid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5) or a variantthereof; wherein a fourth motif of said six motifs comprises the aminoacid sequence CPKISFEP (SEQ ID NO: 6) or a variant thereof; wherein afifth motif of said six motifs comprises the amino acid sequenceFRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variant thereof; and whereina sixth motif of said six motifs comprises the amino acid sequence CSSor a variant thereof, said gp120 of HIV comprising or consisting of (i)the sequence of SEQ ID NO: 2; (ii) the sequence encoded by the sequenceof SEQ ID NO: 1; (iii) a sequence being at least 50% identical to thesequence of SEQ ID NO: 2 or to the sequence encoded by the sequence ofSEQ ID NO: 1; or (iv) a sequence encoded by a sequence being at least50% identical to the sequence of SEQ ID NO: 1, wherein said sequence of(iii) or (iv) comprises or encodes said motifs or variants thereof; and(c) identifying those compounds which interact with at least two aminoacid residues comprised in said six motifs within the 3-dimensionalstructure of said gp120 in (b).

An interaction with at least two amino acid residues comprised in sixmotifs within the 3-dimensional structure of said gp120 of HIV or withina peptidomimetic reflecting the 3-dimensional structure of said gp120 ofHIV is indicative that an inhibitor of the binding of HIV (humanimmunodeficiency virus) glycoprotein (gp)120 to a CD4-receptor or to theintegrin alpha4 beta7 (a4b7) has been identified.

In a further embodiment, the invention relates to a method ofidentifying an inhibitor of the binding of HIV (human immunodeficiencyvirus) glycoprotein (gp)120 to a CD4-receptor or to the integrin alpha4beta7 (a4b7), the method comprising (a) bringing into contact a HIVgp120 or a peptidomimetic reflecting the three-dimensional structure ofsaid gp120 and a test compound; (b) determining whether said testcompound interacts with at least two amino acid residues comprised insix motifs forming a tunnel within the 3-dimensional structure of saidgp120 or within a peptidomimetic reflecting the 3-dimensional structureof said gp120, wherein said interaction between said at least two aminoacid residues and said compound is characterized by an interatomicdistance of less than 8 Angströms, wherein a first motif of said sixmotifs comprises the amino acid sequence DIISLWDQSLKPCVKLT (SEQ ID NO:3) or a variant thereof; wherein a second motif of said six motifscomprises the amino acid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ IDNO: 4) or a variant thereof; wherein a third motif of said six motifscomprises the amino acid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5)or a variant thereof; wherein a fourth motif of said six motifscomprises the amino acid sequence CPKISFEP (SEQ ID NO: 6) or a variantthereof; wherein a fifth motif of said six motifs comprises the aminoacid sequence FRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variantthereof; and wherein a sixth motif of said six motifs comprises theamino acid sequence CSS or a variant thereof, said gp120 of HIVcomprising or consisting of (i) the sequence of SEQ ID NO: 2; (ii) thesequence encoded by the sequence of SEQ ID NO: 1; (iii) a sequence beingat least 50% identical to the sequence of SEQ ID NO: 2 or to thesequence encoded by the sequence of SEQ ID NO: 1; or (iv) a sequenceencoded by a sequence being at least 50% identical to the sequence ofSEQ ID NO: 1, wherein said sequence of (iii) or (iv) comprises orencodes said motifs or variants thereof; and (c) identifying thosecompounds which interact with at least two amino acid residues comprisedin said six motifs forming a tunnel within the 3-dimensional structureof said gp120 in (b). The preferred embodiments described herein beloware also preferred embodiments with regard to this embodiment.

These embodiments relate to a screen for the identification of HIV gp120inhibitors which in turn are suitable as medicaments or lead compoundsfor the development of a medicament. The screen may be implemented invarious ways such as a biochemical screen or a cellular screen. In caseof a cellular screen said “bringing into contact a HIV gp120 or apeptidomimetic reflecting the three-dimensional structure of said gp120and a test compound” may be effected, e.g., by bringing into contact anHIV or a cell producing said virus or said peptidomimetic with a testcompound. Alternatively or additionally, also isolated (recombinantlyproduced) gp120 molecules may be brought into contact with the testcompound. The bringing into contact is performed under conditions whichallow interaction of the test compound to gp120, in case the testcompound is in principle capable of binding as required. Suitableconditions include conditions in liquid phase such as aqueous solutions,preferably buffered solutions. Furthermore, ionic strength may beadjusted, e.g., by the addition of sodium chloride. The concentration ofsodium chloride may be between 0 and 2 M, preferably between 100 and 200mM. Alternatively, sodium chloride is absent from the assay. Forbiological assays in many cases the presence of one or more furthersubstances, including other salts than sodium chloride, trace elements,anti-oxidants, amino acids, vitamins, growth factors, ubiquitousco-factors such as ATP or GTP, is required. Said further substances mayeither be added individually or provided in complex mixtures such as,e.g., serum. These and further accessory substances are well known inthe art as are concentrations suitable for biological assays. Theskilled person is aware of suitable conditions in dependency of theparticular assay format to be used in the method of screening accordingto the invention.

In a further embodiment, the screen may be implemented as a virtualscreen, i.e., the screen may be performed in silico. Virtual screens maybe implemented by computer-based docking of one test compound at a timeinto the allosteric interaction site, preferably binding site, i.e. thetunnel defined above, wherein both the test compound and the interactionsite are represented in silico. Thereby, the interaction position andconformation is calculated. The interaction site may, for example, becomprised in a representation of the entire gp120 molecule or,alternatively, of those parts only which make up the tunnel as definedherein above. Upon completion of docking, the interaction affinityinvolving determining interatomic distances (or equivalently the freeenergy of interaction as defined herein above) is determined based onthe parameters of the computer representation of the involved molecules.A threshold may be chosen such as to select those test compounds whichare candidate high affinity binders. Suitable software packages areknown in the art and include Chemoffice, CNS, CCP4, ADF and Gold (seeabove).

In a preferred embodiment of the method of the invention, saiddetermining in step (b) is effected by X-ray crystallography and/or NMRspectroscopy.

In this embodiment of the invention, the occurrence of an interactioninvolving one or more residues of gp120 is determined by assessingstructural parameters using NMR spectroscopy or X-ray crystallography.These structural parameters may comprise the coordinates of the complexbetween said test compound and gp120. Alternatively, structuralparameters may be determined only to the extent necessary to determinewhether interaction/binding according to the invention, in particularinteraction with one or more residues of gp120 occurs. Examples of thelatter, more selective methods include NMR spectroscopic methodsexploiting the nuclear Overhauser effect or saturation transferdifference (STD). Suitable methods include the recording of NOESY and/orROESY spectra. The required NMR spectra can be obtained in medium tohigh throughput manner, wherein throughput may be further increased byassessing a mixture of ligands, for example 10, 20 or 100 ligands at atime and further analyzing only those mixtures which are found tocomprise one or more binding molecules. Also, means and methods for highthroughput crystallization are available; see, for example, Stevens(Current Opinion in Structural Biology 2000, 10: 558-564) and Kuhn etal. (Current Opinion in Chemical Biology 2002, 6: 704-710).

In a more preferred embodiment, X-ray crystallography comprises (a)generating a crystal of a complex formed by said test compound bound togp120; (b) generating and recording x-ray diffraction data; (c)digitising the data; (d) calculating an electron density map; (e)determining the three-dimensional structure of the crystal components;and (f) storing the crystal coordinates generated on a data carrier.

X-ray diffraction may be performed on a beamline such as the ID29beamline of ESRF, Grenoble or using in-house devices such as a BrukerX8PROTEUM. Data may be further processed with XDS (W. Kabsch, J. Appl.Cryst. 21, 67 (1988)) and refined with CNS (A. T. Brünger et al. ActaCryst. D 54, 905 (1998)). In one alternative, the PROTEUM2 software(Bruker) may be used. Structure can further be solved with, for example,AmoRe (J. Navaza, Acta Crystallogr. A 50, 157 (1994)) and analysed withXfit (D. E. McRee, J. Struct. Biol. 125, 156 (1999)) while structurevalidation may be performed with PROCHECK (R. A. Laskowski, M. W.MacArthur, J. Appl. Crystallogr. 26, 283 (1993)) and WHATCHECK (R. W. W.Hooft, G. Vriend, C. Sander, E. E. Abola, Nature 381, 272 (1996)). Thefinal map containing the atomic coordinates of the constituents of thecrystal may be stored on a data carrier; typically, the data is storedin PDB format or in X_PLOR format, both of which are known to the personskilled in the art. However, crystal coordinates may as well be storedin simple tables or text files.

In another preferred embodiment of the invention, the method furthercomprises the step of (a′) (i) determining whether said test compoundforms a complex with said gp120; and/or (ii) determining whether saidtest compound modulates the activity and/or conformation of said gp120;and/or (iii) determining the cytotoxicity of said test compound; whereinstep (a′) is to be effected after step (a) and prior to step (b), andwherein said determining in step (b) is performed with test compoundsdetermined to bind, to modulate, and/or to be non-cytotoxic in step(a′).

This embodiment provides for filtering a subset of test compoundstesting positive in one or more of the assays of (a′) (i) to (iii). Theadvantage of such filtering is that the subsequent determining whetherthe test compound interacts with one or more residues of gp120 has to bedone only with said test compounds testing positive. Assaying for testcompounds that form complexes with HIV gp120 filter may be advantageouswhen large libraries of compounds are screened for potential inhibitors.Applying the step of filtering out only those test compounds that form acomplex with HIV gp120, limits interaction studies only to thosecompounds instead of the entire library.

Means and methods for identifying complexes formed by interactionpartners, e.g. between a test compound and HIV-1 and/or HIV-2 gp120, arewell known in the art and include assays based on fluorescence such asfluorescence resonance energy transfer (FRET) assays and fluorescencepolarization (FP) assays, immunological assays such as ELISA, surfaceplasmon resonance, isothermal titration calorimetry and FourierTransformed Infrared Spectroscopy (FTIR).

The term “activity of gp120” is meant to refer to the capability ofgp120 to bind to CD4 and/or integrin a4b7 which results in theinitiation of viral entry. Also encompassed is the binding toco-receptors such as, e.g., CXCR5 and CCR5. Assays for assessing gp120activity are discussed further below.

Since the method of identifying an inhibitor according to the inventionis designed to identify compounds binding to the allosteric tunnel ofgp120, an additional or alternative filtering step involves thedetermining whether the test compound modulates or changes theconformation of said gp120. A change in conformation may be determinedby using methods known in the art including the determination ofelectrophoretic or chromatographic mobility and fluorescence-basedmethods. In the latter case, changes in intramolecular distances betweenfluorophors arising from a change of conformation may be determined.

Cytotoxicity may, e.g., be assayed in a cellular assay by determiningthe cellular uptake of neutral red. Only living cells are capable ofneutral red uptake via an active transport mechanism. Hence whenapplying a target compound to cells preferably in varying concentrationsthe cytotoxicity of said target compound may be assessed.

In a more preferred embodiment of the method of the invention, saidactivity is the capability of said gp120 to initiate viral entry of HIVinto mammalian cells. Preferably, the mammalian cell is a human cell. Asdescribed above, activity of gp120 is defined to be its capability tobind to CD4 and the relevant co-receptors as well as integrin a4b7 andinitiate viral entry. Said binding results in a conformational change ofeach of the three gp120 molecules making up the gp120 trimer shieldinggp41. Thereby subsequently an interaction of gp41 with the membrane ofthe target cell is allowed. In other words, the interplay of gp120 andCD4 or integrin a4b7 resulting in the initiation of viral entry isreferred to as activity of gp120. Methods to determine said activity arewell-known in the art and comprise, e.g., the assays described inExample 2 (cell-cell and virus-cell infection assay) determining whethercells have been infected or not.

In another preferred embodiment of the method of the invention, themodelled compound additionally interacts with or the test compound isdetermined to additionally interact with a further motif of HIV gp120 orcorresponding motifs in a peptidomimetic reflecting thethree-dimensional structure of said gp120 molecule.

In accordance with the invention it is preferred that the modelledcompound or the test compound interacts additionally with parts, i.emotifs, of gp120 which are not part of the tunnel. Preferably, saidparts are in the direct vicinity of the tunnel such as, e.g. the bindingsite I of HIV gp120 that has been described herein previously. In otherwords, a compound interacting with the tunnel of gp120 in accordancewith the invention may be (i) one entity that interacts exclusively withthe tunnel or interacts with the tunnel and a part of gp120 other thanthe tunnel. A preferred exemplary motif that can be interacted with by amodelled compound or a test compound in addition to said at least twoamino acid residues comprised in said six motifs, is a motif (externalmotif) comprising the amino acid sequence CRIKQIINMWQEVGKAMYAPPI (SEQ IDNO: 8) of SEQ ID NO: 2 or a variant thereof. Said external motif islocated at an end of the tunnel and as such interaction of a compoundwith said external alone does not result in an inhibition of gp120'sability to change its conformation and as a result inhibit binding toCD4 and/or a4b7. The definitions of “interaction”, “interaction sites”and other definitions relating to motifs described herein also apply tothe interaction of a modelled compound or a test compound with saidexternal motif and to said external motif itself. Preferably, theinteraction site for the external motif is located in the amino acidresidue stretch made up of or at least partially involves amino acidsIKQIINMWQEVGKAMY of the external motif (residues 3-18 of SEQ ID NO: 8)or variants thereof. Also preferred is that the interaction site of theexternal motif comprises or consists of only those amino acid residuesthat have been shown to be most conserved for HIV-1 and HIV-2, such asthe underlined amino acid residues of the external motifCRIKQIINMWQEVGKAMYAPPI (SEQ ID NO: 8) for a gp120 of HIV-1 andCRIKQIINMWQEVGKAMYAPPI (SEQ ID NO: 13) for a gp120 of HIV-2.

Variants of the external motif for gp120 of HIV-1 (SEQ ID NO: 23) canhave the amino acid residue R, S, Y, M, F, G, A, L, I, V, W, P, T, H orN at the first position of said motif; the amino acid residue K, G, S,N, Q, I, T, E, P, H, M, W, L, C, Y, V or D at the second position ofsaid motif; the amino acid residue L, M, V; T, N, R, K, Y, C, S, P, F,H, D or E at the third position of said motif; the amino acid residue R,N, E; I, T, Q, S, G, P, M, Y, L, H, A or D at the fourth position ofsaid motif; the amino acid residue L, H, R, P, K, A, N, E, G, T, M, V orS at the fifth position of said motif; the amino acid residue F, V, L,M, T, Y, N, S, K, A, P, R or D at the sixth position of said motif; theamino acid residue V, M, T, L, A, Y, K, G, E, C, R, S or Q at theseventh position of said motif; the amino acid residue R, H, K, S, D, I,T, Y, G, Q, L, C, M, V, E, P or F at the eighth position of said motif;the amino acid residue R, L, K, T, S, V, I, P, G, Y, H, Q, N, A, C, W orD at the ninth position of said motif; the amino acid residue R, C, G,V, L, M, S, A, P, H, K, Y, N or Q at the 10^(th) position of said motif;the amino acid residue R, I, M, L, H, K, A, E, P, T, G, Y, V, W or S atthe 11^(th) position of said motif; the amino acid residue R, G, K, Q,T, S; A, I, V, D, L, P, H, N, C, M, W or Y at the 12^(th) position ofsaid motif; the amino acid residue A, T, I, G, L, S, E, P, K, R, N, Q, Wor D at the 13^(th) position of said motif; the amino acid residue R, E,A; V, K, Q, T, N, D, P, W or S at the 14^(th) position of said motif;the amino acid residue Q, R, S, T, E, L, P, N, I, H, G, A, V, Y, C, M, Wor D at the 15^(th) position of said motif; the amino acid residue V, G,T, S, P, Q, E, C, W, R, K, Y or E at the 16^(th) position of said motif;the amino acid residue I, T, V, L, K, N, A, F, R, C, S, P, Q, H, Y or Eat the 17^(th) position of said motif; the amino acid residue H, F, C,S, V, D, N, M, A, P, L, E, K, T or G at the 18^(th) position of saidmotif; the amino acid residue P, T, S, N, V, C, G, D, L, I, M or H atthe 19^(th) position of said motif; the amino acid residue S, A, N, L,T, H, R, V, K, D or Q at the 20^(th) position of said motif; the aminoacid residue S, L, A, T, F, R, H, G, Y, N or Q at the 21^(th) positionof said motif; and/or the amino acid residue V, T, R, L, F, M, N, P, H,A, K, G, W, Y or D at the 22^(nd) position of said motif, wherein C ofsaid motif takes the first position and I takes the 22^(nd) position ofthe external motif.

Variants of the external motif for gp120 of HIV-2 (SEQ ID NO: 24) canhave the amino acid residue R or Y at the first position of said motif;the amino acid residue H or Q at the second position of said motif; theamino acid residue at the third position of said motif is not variant;the amino acid residue R, P or E at the fourth position of said motif;the amino acid residue at the fifth position of said motif is notvariant; the amino acid residue V or K at the sixth position of saidmotif; the amino acid residue V at the seventh position of said motif;the amino acid residue S at the eighth position of said motif; the aminoacid residue T, A or I at the ninth position of said motif; the aminoacid residue R at the 10^(th) position of said motif; the amino acidresidue H or R at the 11^(th) position of said motif; the amino acidresidue K, R or N at the 12^(th) position of said motif; the amino acidresidue A, I or S at the 13^(th) position of said motif; the amino acidresidue W, R, E at the 14^(th) position of said motif; the amino acidresidue Q, R, I, V, N, T, L or E at the 15^(th) position of said motif;the amino acid residue N, H, Y, R or K at the 16^(th) position of saidmotif; the amino acid residue V, I, A, L or Y at the 17^(th) position ofsaid motif; the amino acid residue I at the 18^(th) position of saidmotif; the amino acid residue L or F at the 19^(th) position of saidmotif; the amino acid residue A or L at the 20^(th) position of saidmotif; the amino acid residue at the 21^(st) position of said motif isnot variant; and/or the amino acid residue R or K at the 22^(nd)position of said motif, wherein C of said motif takes the first positionand I takes the 22^(nd) position of the external motif.

Also envisaged is linking of a modeled/synthesized compound or a testcompound once designed/synthesized or identified in accordance with themethod of the invention to a known gp120 binding compound notinteracting with the tunnel or alternatively to a modelled compound or atest compound that interacts with gp120 other than to the tunnel. Inother words, such compounds interacting with the tunnel of gp120 may becomposed of two entities linked to each other, wherein one entityinteracts with the tunnel and the other entity interacts with said othermotif or part of gp120. A corresponding compound may be a bipartitecompound or one of the two entities can correspond to a discretecompound that is linked to another compound, i.e. the second entity. Thecombination of interaction with the tunnel and other parts of gp120 incontrast to exclusively interacting with the tunnel provides theadvantage of more flexibility as regards designing modelled compounds oridentifying test compounds in view of increasing binding affinities andlikelihood of said compounds at least partially entering the tunnel.

In a further preferred embodiment of the method of the invention,molecular modeling starts from a compound selected from a compound ofthe formula (1) and (2) or a salt or solvate thereof, or the testcompound to be screened in accordance with the method of the inventiondescribed herein is selected from a compound of the formula (1) and (2)or a salt or solvate thereof:

As demonstrated in the Example section the above compounds of formula(1) and (2) have been shown to be capable of inhibiting the binding ofCD4 or integrin a4b7 to gp120 through interaction with the noveltargeting site, i.e. the tunnel. Accordingly, molecular modeling may bebased on the structural information of the compounds of formula (1) and(2). Such molecular modeling starting from said compounds may lead toimproved compounds as described below. The compound of formula (1)corresponds to the compound termed “T5379534” in example 2 and 3 (andFIGS. 35 and 37) while the compound of formula (2) corresponds to thecompound termed “T0520-5895” in example 2 and 3 (and FIG. 34 and FIG.37).

The compound of formula (1) interacts with sequence motifs 1, 2, 3, 5within the 3-dimensional structure of the gp120 used for experimentationand with the external motif described herein above. Specifically, saidcompound has been demonstrated to interact with the second “I”, “W” and“KLT” of said first motif (residues 15-17 of SEQ ID NO: 3) at 12Angström, with the second “I” and “W” of said first motif at 8 Angström;with “VVSTQ” of the second motif (residues 14-18 of SEQ ID NO: 4) at 12Angström, with “VST” of the second motif (residues 15-17 of SEQ ID NO:4) at 8 Angström and “T” of said interaction site “VVSTQ” of the secondmotif (residues 14-18 of SEQ ID NO: 4) at 5 Angström; with“GGDPEIVMHSFN” (residues 2-13 of SEQ ID NO: 5) and the first “F” and the“Y” within the sequence “GGEFFYCN” of the third motif (residues 15-22 ofSEQ ID NO: 5) at 12 Angström; with “GDPEIVMHSFN” (residues 3-13 of SEQID NO: 5) and the first “F” and the “Y” within the sequence “GGEFFYCN”of the third motif (residues 15-22 of SEQ ID NO: 5) at 8 Angström; with“D”, “EI” and “SFN” within the sequence of “GGDPEIVMHSFN” (residues 2-13of SEQ ID NO: 5) and the first “F” and the “Y” within the sequence“GGEFFYCN” of the third motif (residues 15-22 of SEQ ID NO: 5) at 5Angström; with “IINMWQEVGKA” within the external motif (residues 6-16 ofSEQ ID NO: 8) at 12 Angström; with “IINMWQEVGK” within the externalmotif (residues 6-15 of SEQ ID NO: 8) at 8 Angström; with “INMWQEV”within the external motif (residues 7-13 of SEQ ID NO: 8) at 5 Angström;with “GGGDMRDNW” within the fifth motif (residues 4-12 of SEQ ID NO: 7)at 12 Angström, with “GGDMR” within the fifth motif (residues 5-9 of SEQID NO: 7) at 8 Angström, and with “GDMR” within the fifth motif(residues 6-9 of SEQ ID NO: 7) at 5 Angström.

The compound of formula (2) interacts with the same sequence motifs asthe above compound within the 3-dimensional structure of the gp120 usedfor experimentation. Specifically, said compound has been demonstratedto interact with the second “I” and “W” of said first motif (SEQ ID NO:3) at 12 Angström, with the second “I” and “W” of said first motif (SEQID NO: 3) at 8 Angström; with the “W” of said first motif at 5 Angström;with “VVSTQ” of the second motif (residues 14-18 of SEQ ID NO: 4) at 12Angström, with “VST” of the second motif (residues 15-17 of SEQ ID NO:4) at 8 Angström, “V” and “T” of said interaction site “VVSTQ” of thesecond motif (residues 14-18 of SEQ ID NO: 4) at 5 Angström; with“GGDPEIVMHSFN” (residues 2-13 of SEQ ID NO: 5) and the first “F” and the“Y” within the sequence “GGEFFYCN” of the third motif (residues 15-22 ofSEQ ID NO: 5) at 12 Angström; with “GDPEIVMHSFN” (residues 3-15 of ofSEQ ID NO: 5) and the first “F” and the “Y” within the sequence“GGEFFYCN” of the third motif (residues 15-22 of SEQ ID NO: 5) at 8Angström; with “D”, “EI” and “SFN” within the sequence of “GGDPEIVMHSFN”(residues 2-13 of SEQ ID NO: 5) and the first “F” and the “Y” within thesequence “GGEFFYCN” of the third motif (residues 15-22 of SEQ ID NO: 5)at 5 Angström; with “IINMWQEVGKA” within the external motif (residues6-16 of SEQ ID NO: 8) at 12 Angström; with “IINMWQEVGK” within theexternal motif (residues 6-15 of SEQ ID NO: 8) at 8 Angström; with“INMWQEV” within the external motif (residues 7-13 of SEQ ID NO: 8) at 5Angström; with “GGGDMRDNW” within the fifth motif (residues 4-12 of SEQID NO: 7) at 12 Angström, with “GGDMR” (residues 5-9 of SEQ ID NO: 7)and “N” in the sequence of “GGGDMRDNW” within the fifth motif (residues4-12 of SEQ ID NO: 7) at 8 Angström, and with “GDMR” within the fifthmotif (residues 6-9 of SEQ ID NO: 7) at 5 Angström. In the case ofvariant motifs forming a tunnel within the 3-dimensional structure ofgp120 molecule, said compounds of formula (1) and (2) may interact withless than motifs 1, 2, 3, 5 and the external motif and/or with differentamino acid residues within said motifs and still be considered aninhibitor in accordance with the invention, provided that the inhibitorbinds to at least two amino acid residues comprised in said motifsforming the tunnel.

A further embodiment of the invention relates to a compound according toformula (1) or (2) as defined herein above. Preferably, the compoundaccording to formula (1) or (2) is used as a medicament and/or as a leadcompound to further improve the pharmaceutical characteristics of thecompound. The skilled person is well-aware of the different formulationsa medicament may be made of such as, e.g., the formulations as describedherein below.

In another embodiment the invention relates to a method of decreasingthermal motion of a tunnel within gp120 comprising contacting said gp120with a compound that interacts with at least two amino acid residuescomprised in six motifs within the 3-dimensional structure of said gp120of HIV or within a peptidomimetic reflecting the 3-dimensional structureof said gp120 of HIV, wherein said interaction between said at least twoamino acid residues and said compound is characterized by an interatomicdistance of less than 8 Angströms, wherein a first motif of said sixmotifs comprises the amino acid sequence DIISLWDQSLKPCVKLT (SEQ ID NO:3) or a variant thereof; wherein a second motif of said six motifscomprises the amino acid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ IDNO: 4) or a variant thereof; wherein a third motif of said six motifscomprises the amino acid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5)or a variant thereof; wherein a fourth motif of said six motifscomprises the amino acid sequence CPKISFEP (SEQ ID NO: 6) or a variantthereof; wherein a fifth motif of said five motifs comprises the aminoacid sequence FRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variantthereof; and wherein a sixth motif of said six motifs comprises theamino acid sequence CSS or a variant thereof, said gp120 of HIVcomprising or consisting of (i) the sequence of SEQ ID NO: 2; (ii) thesequence encoded by the sequence of SEQ ID NO: 1; (iii) a sequence beingat least 50% identical to the sequence of SEQ ID NO: 2 or to thesequence encoded by the sequence of SEQ ID NO: 1; or (iv) a sequenceencoded by a sequence being at least 50% identical to the sequence ofSEQ ID NO: 1, wherein said sequence of (iii) or (iv) comprises orencodes said motifs or variants thereof.

An interaction with at least two amino acid residues comprised in sixmotifs within the 3-dimensional structure of said gp120 of HIV or withina peptidomimetic reflecting the 3-dimensional structure of said gp120 ofHIV is indicative that a decrease of the thermal motion of said tunnelwithin gp120 has been achieved.

In a further embodiment, the invention relates to a method of decreasingthermal motion of a tunnel within gp120 comprising contacting said gp120with a compound that interacts with at least two amino acid residuescomprised in six motifs forming a tunnel within the 3-dimensionalstructure of said gp120 of HIV or within a peptidomimetic reflecting the3-dimensional structure of said gp120 of HIV, wherein said interactionbetween said at least two amino acid residues and said compound ischaracterized by an interatomic distance of less than 8 Angströms,wherein a first motif of said six motifs comprises the amino acidsequence DIISLWDQSLKPCVKLT (SEQ ID NO: 3) or a variant thereof; whereina second motif of said six motifs comprises the amino acid sequenceNVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ ID NO: 4) or a variant thereof; whereina third motif of said six motifs comprises the amino acid sequenceSGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5) or a variant thereof; wherein afourth motif of said six motifs comprises the amino acid sequenceCPKISFEP (SEQ ID NO: 6) or a variant thereof; wherein a fifth motif ofsaid five motifs comprises the amino acid sequenceFRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variant thereof; and whereina sixth motif of said six motifs comprises the amino acid sequence CSSor a variant thereof, said gp120 of HIV comprising or consisting of (i)the sequence of SEQ ID NO: 2; (ii) the sequence encoded by the sequenceof SEQ ID NO: 1; (iii) a sequence being at least 50% identical to thesequence of SEQ ID NO: 2 or to the sequence encoded by the sequence ofSEQ ID NO: 1; or (iv) a sequence encoded by a sequence being at least50% identical to the sequence of SEQ ID NO: 1, wherein said sequence of(iii) or (iv) comprises or encodes said motifs or variants thereof.

The term “thermal motion” is well-known in the art and has the samemeaning in accordance with the invention. Briefly, thermal motionrelates to the random motion of molecules that results from their beingin thermal equilibrium at a particular temperature. In general, thermalmotions are faster at higher temperatures and for lower mass particles.In accordance with the present invention, decreasing thermal motion ofthe tunnel will inhibit the flexibility of gp120 which results in theincapability of changing the conformation of gp120 required for bindingto CD4 or to the integrin a4b7. In other words, the capability of gp120to undergo a conformational change is dependent on the degree of thermalmotion of said tunnel within gp120 as defined herein. The term“decreasing” means in accordance with the present invention lowering orcompletely abolishing said thermal motion. In preferred embodiments, adecrease refers to a reduction in thermal motion of at least (for eachvalue) 10, 20, 30, 40, more preferred at least 50, 60, 70, 80, 90, 95,98 and most preferred at least 99% as well as a reduction of 100%, i.e.completely abolishing thermal motion. Also preferred, thermal motiondrops to less than 10⁻², less than 10⁻³, less than 10⁻⁴ or less than10⁻⁵ times the activity compared to the activity in the absence of thecompound.

In another embodiment, the invention relates to a compound capable ofinteracting with at least two amino acid residues comprised in sixmotifs within the 3-dimensional structure of said gp120 of HIV or withina peptidomimetic reflecting the 3-dimensional structure of said gp120 ofHIV, wherein said interaction between said at least two amino acidresidues and said compound is characterized by an interatomic distanceof less than 8 Angströms, wherein a first motif of said six motifscomprises the amino acid sequence DIISLWDQSLKPCVKLT (SEQ ID NO: 3) or avariant thereof; wherein a second motif of said six motifs comprises theamino acid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ ID NO: 4) or avariant thereof; wherein a third motif of said six motifs comprises theamino acid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5) or a variantthereof; wherein a fourth motif of said six motifs comprises the aminoacid sequence CPKISFEP (SEQ ID NO: 6) or a variant thereof; wherein afifth motif of said five motifs comprises the amino acid sequenceFRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variant thereof; and whereina sixth motif of said six motifs comprises the amino acid sequence CSSor a variant thereof, said gp120 of HIV comprising or consisting of (i)the sequence of SEQ ID NO: 2; (ii) the sequence encoded by the sequenceof SEQ ID NO: 1; (iii) a sequence being at least 50% identical to thesequence of SEQ ID NO: 2 or to the sequence encoded by the sequence ofSEQ ID NO: 1; or (iv) a sequence encoded by a sequence being at least50% identical to the sequence of SEQ ID NO: 1, wherein said sequence of(iii) or (iv) comprises or encodes said motifs or variants thereof.

The skilled person is in the position to identify compounds that caninteract with said at least two amino acid residues comprised in sixsequence motifs within the three-dimensional structure of said gp120 ofHIV, for example in accordance with methods described herein above, i.e.in silico methods such as used in molecular modeling. Briefly, thetertiary structure of a compound is determined by methods well-known inthe art or obtained otherwise, e.g. from a database comprising data ofthe tertiary structure of compounds, and then used in an in silicomethod as described above that enable the analysis whether the compoundto be analysed can interact with said at least two amino acid residuecomprised in six motifs within the 3-dimensional structure of gp120 ofHIV. It is understood in accordance with the invention that a compoundas defined herein above can be used as an inhibitor of the binding ofCD4-receptor or integrin a4b7 to gp120 of HIV. Therefore, it is alsounderstood that a compound as defined herein can be used for theprevention and/or treatment of a HIV-infection and/or a diseaseassociated with a HIV-infection as detailed below.

In another embodiment, the invention relates a method of inhibiting thebinding of HIV (human immunodeficiency virus) glycoprotein (gp)120 tothe integrin alpha4 beta? (a4b7), the method comprising the step ofinhibiting a conformational change of said gp120 taking place uponbinding to said integrin a4b7, wherein inhibition of said conformationalchange inhibits the binding of HIV-1 or HIV-2 gp120 to said integrin.

Without wishing to be bound by specific theory and as explained hereinabove, it is expected on the basis of experimental evidence (see example3) that gp120 undergoes a conformational change to be able to bind tothe integrin a4b7. Therefore, inhibiting the conformational change thatgp120 must undergo to be able to bind to said integrin will lead to theinhibition of said binding of gp120 to integrin a4b7.

In a preferred embodiment of the invention, the inhibition is effectedby decreasing the thermal motion of a tunnel within said gp120, andwherein the 3-dimensional structure of said tunnel within the3-dimensional structure of said gp120 is formed by six motifs, wherein afirst motif of said six motifs comprises the amino acid sequenceDIISLWDQSLKPCVKLT (SEQ ID NO: 3) or a variant thereof; wherein a secondmotif of said six motifs comprises the amino acid sequenceNVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ ID NO: 4) or a variant thereof; whereina third motif of said six motifs comprises the amino acid sequenceSGGDPEIVMHSFNCGGEFFYCN (SEQ ID NO: 5) or a variant thereof; wherein afourth motif of said six motifs comprises the amino acid sequenceCPKISFEP (SEQ ID NO: 6) or a variant thereof; and wherein a fifth motifof said six motifs comprises the amino acid sequenceFRPGGGDMRDNWRSELYKYKVV (SEQ ID NO: 7) or a variant thereof; and whereina sixth motif of said six motifs comprises the amino acid sequence CSSor a variant thereof, said gp120 of HIV comprising or consisting of (i)the sequence of SEQ ID NO: 2; (ii) the sequence encoded by the sequenceof SEQ ID NO: 1; (iii) a sequence being at least 50% identical to thesequence of SEQ ID NO: 2 or to the sequence encoded by the sequence ofSEQ ID NO: 1; or (iv) a sequence encoded by a sequence being at least50% identical to the sequence of SEQ ID NO: 1, wherein said sequence of(iii) or (iv) comprises or encodes said motifs or variants thereof.

In a more preferred embodiment of the method of the invention, thedecrease in thermal motion is effected according to the method ofdecreasing thermal motion of a tunnel within gp120 of HIV defined hereinabove.

In another more preferred embodiment of the invention, the method isfurther effecting the inhibition of the binding of HIV gp120 to aCD4-receptor.

As has been described herein above in detail before, the capability ofgp120 to bind to CD4 depends on the structural flexibility of gp120which in turn depends on the flexibility of the tunnel. Said flexibilityof the tunnel is dependent from the thermal motion of the tunnel. Hence,decreasing the thermal motion of said tunnel within gp120 of HIV willlead to the inhibition of the conformational change necessary for gp120to bind to CD4.

In a further embodiment, the invention relates to the use of a compoundselected from compounds of the formula (1) or (2) as defined hereinabove or a salt or solvate thereof as a lead compound in the developmentof an inhibitor of the binding of a HIV gp120 to a CD4-receptor orintegrin alpha4 beta? (a4b7).

The term “lead compound” is known in the art and refers to a compoundproviding a starting point for developing a pharmaceutically activeagent. Generally, said pharmaceutically active agent is different from,preferably optimized as compared to the lead compound. In other words,the development of a lead compound preferably involves the optimizationof the pharmacological properties of said lead compound. The term “leadcompound” therefore refers also to a compound that is analyzed only withregard to the parts of the compound that are capable of interacting withat least two amino acid residues comprised in said six motifs within the3-dimensional structure of gp120. In other words, one can devise othercompounds on the basis of lead compounds that may not or may onlypartially be structurally related to the lead compound but that arefunctionally related, i.e. show the desired activity. In accordance withthe present invention a functionally related compound is a compound thatinteracts with at least two amino acid residues comprised in said sixmotifs or variants thereof within the 3-dimensional structure of gp120.It is understood that said functional relationship includes thecapability of inhibiting the binding of gp120 to CD4 and/or integrina4b7.

Methods for the optimization of the pharmacological properties ofcompounds identified in screens, generally referred to as leadcompounds, are known in the art and comprise a method of modifying acompound identified as a lead compound to achieve: (i) modified site ofaction, spectrum of activity, organ specificity, and/or (ii) improvedpotency, and/or (iii) decreased toxicity (improved therapeutic index),and/or (iv) decreased side effects, and/or (v) modified onset oftherapeutic action, duration of effect, and/or (vi) modifiedpharmacokinetic parameters (resorption, distribution, metabolism andexcretion), and/or (vii) modified physico-chemical parameters(solubility, hygroscopicity, color, taste, odor, stability, state),and/or (viii) improved general specificity, organ/tissue specificity,and/or (ix) optimized application form and route by (i) esterificationof carboxyl groups, or (ii) esterification of hydroxyl groups withcarboxylic acids, or (iii) esterification of hydroxyl groups to, e.g.phosphates, pyrophosphates or sulfates or hemi-succinates, or (iv)formation of pharmaceutically acceptable salts, or (v) formation ofpharmaceutically acceptable complexes, or (vi) synthesis ofpharmacologically active polymers, or (vii) introduction of hydrophilicmoieties, or (viii) introduction/exchange of substituents on aromates orside chains, change of substituent pattern, or (ix) modification byintroduction of isosteric or bioisosteric moieties, or (x) synthesis ofhomologous compounds, or (xi) introduction of branched side chains, or(xii) conversion of alkyl substituents to cyclic analogues, or (xiii)derivatisation of hydroxyl group to ketales, acetales, or (xiv)N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannichbases, imines, or (xvi) transformation of ketones or aldehydes to Schiffs bases, oximes, acetales, ketales, enolesters, oxazolidines,thiazolidines or combinations thereof.

The various steps recited above are generally known in the art. Theyinclude or rely on quantitative structure-action relationship (QSAR)analyses (Kubinyi, “Hausch-Analysis and Related Approaches”, VCH Verlag,Weinheim, 1992), combinatorial biochemistry, classical chemistry andothers (see, for example, Holzgrabe and Bechtold, Deutsche ApothekerZeitung 140(8), 813-823, 2000).

Another embodiment of the invention relates to a pharmaceuticalcomposition comprising one or more of the compounds selected from theformula (1) or (2) as defined herein above or salts or solvates orfunctional derivatives thereof or a compound as defined herein above.While said compounds of formula (1) or (2) may be used as a leadcompound, which includes as mentioned previously optimizing the compound(e.g., to increase its bioavailability), said compounds have shown sucheffectiveness against HIV infection in tests that they can be formulatedinto a pharmaceutical composition to be used in a treatment regimen of,e.g., an HIV-infection and/or a disease associated with anHIV-infection, optionally with further active ingredients to this end.

Equally envisaged is a pharmaceutical composition comprising one or moreof the inhibitors or salts or solvates or functional derivatives thereofdesigned or identified according to the methods described herein above.

The pharmaceutical composition may further comprise pharmaceuticallyexcipients. Pharmaceutically acceptable excipients that may be used inthe formulation of the pharmaceutical compositions may comprisecarriers, vehicles, diluents, solvents such as monohydric alcohols suchas ethanol, isopropanol and polyhydric alcohols such as glycols andedible oils such as soybean oil, coconut oil, olive oil, safflower oilcottonseed oil, oily esters such as ethyl oleate, isopropyl myristate;binders, adjuvants, solubilizers, thickening agents, stabilizers,disintegrants, glidants, lubricating agents, buffering agents,emulsifiers, wetting agents, suspending agents, sweetening agents,colourants, flavours, coating agents, preservatives, antioxidants,processing agents, drug delivery modifiers and enhancers such as calciumphosphate, magnesium state, talc, monosaccharides, disaccharides,starch, gelatine, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone,low melting waxes, ion exchange resins. Other suitable pharmaceuticallyacceptable excipients are described in Remington's PharmaceuticalSciences, 15th Ed., Mack Publishing Co., New Jersey (1991). Compositionscomprising such carriers can be formulated by well known conventionalmethods. These pharmaceutical compositions can be administered to thesubject at a suitable dose. Administration of the suitable compositionsmay be effected by different ways, e.g., by intravenous,intraperitoneal, subcutaneous, intramuscular, topical, intradermal,intranasal or intrabronchial administration. It is particularlypreferred that said administration is carried out by injection and/ordelivery, e.g., to a site in the pancreas or into a brain artery ordirectly into brain tissue. The compositions may also be administereddirectly to the target site, e.g., by biolistic delivery to an externalor internal target site, like the pancreas or brain. The dosage regimenwill be determined by the attending physician and clinical factors. Asis well known in the medical arts, dosages for any one patient dependsupon many factors, including the patient's size, body surface area, age,the particular compound to be administered, sex, time and route ofadministration, general health, individual response of the patient to betreated, severity of the disease to be treated, the activity andbioavailability of the particular compound applied and other drugs beingadministered concurrently. Pharmaceutically active matter may be presentin amounts between 1 ng and 10 mg/kg body weight per dose; however,doses below or above this exemplary range are envisioned, especiallyconsidering the aforementioned factors. If the regimen is a continuousinfusion, it is preferably in the range of 1 μg to 10 mg units perkilogram of body weight per minute.

The pharmaceutical compositions of the invention can be produced in amanner known per se to the skilled person or as described, for example,in Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co.,New Jersey (1991).

A further embodiment of the invention relates to a compound selectedfrom the formula (1) or (2) as defined herein above or salts or solvatesor functional derivatives thereof or a compound as defined herein abovefor use in preventing or treating a HIV-infection and/or a diseaseassociated with a HIV-infection.

As used herein, the term “HIV-infection” generally encompasses infectionof a host, particularly a human host, by the human immunodeficiencyvirus (HIV) family of retroviruses including, but not limited to, HIV-1,HIV-2 (previously also known as HTLV-III/LAV/ARV, LAV-1, LAV-2). “HIV”can be used herein to refer to any strains, forms, subtypes, classes andvariations in the HIV family. Thus, “treatment” of a HIV-infectionand/or a disease associated with a HIV-infection will encompass thetreatment of a person who is a carrier of any of the HIV family ofretroviruses or a person who is diagnosed of active AIDS, as well as thetreatment or prophylaxis of AIDS-related conditions in such persons.AIDS is also an example of a disease associated with an HIV-infection.The skilled person is well-aware of the pathology of AIDS includinginitiation, progression and clinical outcomes. A carrier of HIV may beidentified by any method known in the art. For example, a person can beidentified as an HIV carrier on the basis that the person is anti-HIVantibody positive, or is HIV-positive, or has symptoms of AIDS. That is,“treating HIV-infection” should be understood as treating a patient whois at any one of the several stages of HIV infection progression, which,for example, include acute primary infection syndrome (which can beasymptomatic or associated with an influenza-like illness with fevers,malaise, diarrhea and neurologic symptoms such as headache),asymptomatic infection (which is the long latent period with a gradualdecline in the number of circulating CD4 positive T cells), and AIDS(which is defined by more serious AIDS-defining illnesses and/or adecline in the circulating CD4 cell count to below a level that iscompatible with effective immune function). In addition, “preventing ortreating of a disease associated with an HIV-infection” will alsoencompass treating suspected infection by HIV after suspected pastexposure to HIV by e.g., contact with HIV-contaminated blood, as aresult of blood transfusion, exchange of body fluids, “unsafe” sex withan infected person, accidental needle stick, receiving a tattoo oracupuncture with contaminated instruments, or transmission of the virusfrom a mother to a baby during pregnancy, delivery or shortlythereafter. The term “preventing” also encompasses treating a person whohas not been diagnosed as having a HIV infection but is believed to beat risk of infection by HIV. Diseases associated with an HIV-infectioncan generally be treated by eradicating the primary cause thereof,optionally in conjunction with medicaments known in the art that areregistered for the treatment of such secondary causes.

The skilled person is well-aware of the pathology of a HIV-infection anddiseases associated with a HIV-infection and hence is in the position todevise a therapy according to general principles known in the art anddescribed, for example, elsewhere herein.

The impact of a drug that inhibits new infection of CD4⁺ cells will leadto a recuperation of said cell population and hence restore thepatient's immune system, i.e. treat AIDS or prevent the outbreak ofAIDS. The same is, of course, also true for a HIV infection that has notyet resulted in AIDS. As mentioned, said recuperation of the CD4⁺ cellpopulation has a beneficial effect also on the fight against secondaryinfections like, e.g., recurring viral infections and bacterialinfections, that characterize the medical condition AIDS and are mostlyresponsible for the death of AIDS patients. The same applies to otherembodiments relating to HIV infection and HIV-associated diseases inthis specification. The usefulness to inhibit gp120 to CD4 binding hasbeen described and discussed herein above. In particular, Example 2demonstrates the potent effect of compounds that act as inhibitors ofthe binding of gp120 to CD4 in accordance with the invention. Equallyenvisaged is an inhibitor or salts or solvates or functional derivativesthereof designed or identified according to the methods described hereinabove for use in preventing or treating a HIV-infection and/or a diseaseassociated with a HIV-infection.

Examples 2 and 3 provide proof of the in vitro activity of saidexemplary compounds of formula (1) and (2) as defined herein above ofthe invention in the inhibition of viral entry and thereby of HIVreplication. As shown in FIGS. 34 and 35, said compounds interact withmotifs in the tunnel.

Accordingly, the invention also relates in an embodiment to a method ofpreventing or treating a HIV-infection and/or a disease associated witha HIV-infection by administering an effective dose of a compoundselected from the formula (1) and/or (2) or inhibitors designed oridentified according to the methods described herein above or salts orsolvates or functional derivatives thereof to a patient in need thereof.

THE FIGURES SHOW:

FIG. 1: Overlap of the residue sets of binding site I and binding siteII.

FIG. 2: Residues of binding site I are located in a large cavity.

FIG. 3: PHE43 binding pocket. CD4 is depicted as a ribbon model with thePHE43 residue differently designed reaching into the pocket.

FIG. 4: The tunnel connecting the two binding sites I and II. The figureshows that binding site I and binding site II are the two sides of afurther active site buried within gp120.

FIG. 5: A This figure represents the cut-in view inside the gp120protein of the tunnel connecting the binding sites I and II with a modelligand resting in the tunnel.

B: Active sites I and II volume fluctuations along the MD trajectory. Tocalculate the active site volume the active site was filled by spheresgenerated by Ligand module of QUANTUM software and calculated the numberof spheres in each protein conformation (frame) The volume of Site I isindicated by the lower line and the volume of Site II is indicated bythe upper line (both are labeled).

FIG. 6: Overlapping-PCR strategy utilized for generating each one of thetwo mutants of example 2.

FIG. 7: Principle of dual-enhancement of Cell-infection for Phenotypingresistance (deCIPhR) as used in example 2.

FIG. 8: Principle of a Virus to Cell-infection system as used, e.g., inexample 2.

FIG. 9: Complete dose-response curves for T5379534 and T0520-5895 andreferences T20 and EFV on pNL4-3, pNL-Bal and pNL-AD87 HIV-1 viruses.Percentage inhibition of viral replication (average of triplicate) areplotted as a function of compound concentration (log scale). Each curveis identified by an arrow.

Tables below graphs report observed values and calculated inhibitionparameters (see paragraph 4 of example 2). Error bars are omitted forclarity.

FIG. 10: Complete dose-response curves for T5379534 and T0520-5895 andreferences T20 and EFV on pNL4-3 HIV-1 virus in virus-to-cell infectionformat. Percentage inhibition of viral replication (average oftriplicate) are plotted as a function of compound concentration (logscale).

Tables below graphs report observed values and calculated inhibitionparameters (see paragraph 4 of example 2). Error bars are omitted forclarity.

FIG. 11: Complete dose-response curves for T5379534 and T0520-5895 andreferences T20 and EFV on “G-I-A” mutant. Percentage inhibition of viralreplication (average of triplicate) are plotted as a function ofcompound concentration (log scale). Tables below graphs report observedvalues and calculated inhibition parameters (see paragraph 4 of example2).

FIG. 12: Complete dose-response curves for T5379534 & T0520-5895 andreferences T20 and EFV on “D-I-A” mutant. Percentage inhibition of viralreplication (average of triplicate) are plotted as a function ofcompound concentration (log scale). Tables below graphs report observedvalues and calculated inhibition parameters (see paragraph 4 of example2).

FIG. 13: Structure of gp120 and location of the tunnel. A Model ligandplaced in a tunnel gate. To the right of said tunnel gate is the othertunnel gate. B-D Alternate views of gp120 and the tunnel within gp120,wherein the model ligand in the tunnel.

FIG. 14: A-F Alternate views of the tunnel with the motifs 1, 2, 3 and 5of the tunnel and the external motif.

FIG. 15: A Amino acid sequence of gp120 of HIV-1 (residues 6-58 of SEQID NO: 2) comprised in a first motif (Bold amino acid residues andsequence that is flanked by said bold residues). “Conserv” describes theconservation index of the amino acid at said position. A conservationindex of 9 means that 90% to 99,99%, and index of 8 means that 80% to89,99% (and so forth for an index of 7, 6, 5, 4, 3, 2, 1 or 0) of theHIV-1 variants have the amino acid residue specified in the sequence“initial” at this position in the motif, wherein “initial” is the aminoacid sequence of the HIV-1 strain taken for analysis (SEQ ID NO: 2). Thefirst line “Result” (from top to bottom) depicts the consensus sequenceof all HIV-1 variants analysed (˜80,000). The remaining “Result” linesshow the likelihood of variant amino acids replacing the amino acids ofthe motif from top to bottom (top: most likely, bottom: least likely).The same holds true for FIGS. 16 to 19 for HIV-1 and FIGS. 23 to 25 forHIV-2, mutatis mutandis. FIG. 15A discloses “SLKPCVKL” as residues 9-16of SEQ ID NO: 3. B Model showing the tunnel in relation to the motif(ribbon model).

FIG. 16: A Amino acid sequence of gp120 of HIV-1 (residues 85-143 of SEQID NO: 2) comprised in a second motif. FIG. 16A discloses “VVSTQ” asresidues 14-18 of SEQ ID NO: 4. B Model showing the tunnel in relationto the motif (ribbon model).

FIG. 17: A Amino acid sequence of gp120 of HIV-1 (residues 205-262 ofSEQ ID NO: 2) comprised in a third motif. FIG. 17A discloses“DPEIVMHSFNC” as residues 4-14 of SEQ ID NO: 5, “GEFFYC” as residues16-21 of SEQ ID NO: 5 and “EFFYC” as residues 17-21 of SEQ ID NO: 5. BModel showing the tunnel in relation to the motif (ribbon model).

FIG. 18: A Amino acid sequence of gp120 of HIV-1 (residues 252-312 ofSEQ ID NO: 2) comprised in the external motif. FIG. 18 discloses“IKQIINMWQEVGKAMY” as residues 3-18 of SEQ ID NO: 8, “KQIINMWQEVGKAMY”as residues 4-18 of SEQ ID NO: 8 and “INMWQ” as residues 7-11 of SEQ IDNO: 8. B Model showing the tunnel in relation to the motif (ribbonmodel).

FIG. 19: A Amino acid sequence of gp120 of HIV-1 (residues 296-344 ofSEQ ID NO: 2) comprised in a fifth motif. FIG. 19 discloses “GGDMR” asresidues 5-9 of SEQ ID NO: 7 and “GGDM” as residues 5-8 of SEQ ID NO: 7.B Model showing the tunnel in relation to the motif (ribbon model).

FIG. 20: Model of the tunnel within HIV-1 gp120 with motifs 1, 2, 3, 5and the external motif as ribbon model.

FIG. 21: Alternate views of the tunnel and the motifs 1, 2, 3, 5 and theexternal motif making up the tunnel (ribbon model).

FIG. 22: Comparison of motifs 1, 2, 3, and 5 forming the tunnel withingp120 and the external motif of HIV-1 (A: left picture) and HIV_2 (A:right picture). Alternate views of said comparison are shown in B (leftpicture motifs of HIV-1 and right picture motifs of HIV-2)

FIG. 23: A Amino acid sequence of gp120 of HIV-2 (SEQ ID NO: 26)comprised in a first motif (for sequence of said motif see descriptionherein above). FIG. 23 A discloses “SLKPCVKL” as residues 9-16 of SEQ IDNO: 9. B Amino acid sequence of gp120 of HIV-2 (SEQ ID NO: 27).comprised in a second motif (for sequence of said motif see descriptionherein above). FIG. 23 B discloses “VVSTQ” as residues 14-18 of SEQ IDNO: 10.

FIG. 24: A Amino acid sequence of gp120 of HIV-2 (SEQ ID NO: 28)comprised in a third motif (for sequence of said motif see descriptionherein above). FIG. 24A discloses “DPEIVMHSFNC” as residues 4-14 of SEQID NO: 11, “GEFFYC” as residues 16-21 of SEQ ID NO: 11 and residues“EFFYC” as residues 17-21 of SEQ ID NO: 11. B Amino acid sequence ofgp120 of HIV-2 (SEQ ID NO: 29). comprised in the external motif (forsequence of said motif see description herein above). FIG. 24B discloses“IKQIINMWQEVGKAMY” as residues 3-18 of SEQ ID NO: 13, “KQIINMWQEVGKAMY”as residues 4-18 of SEQ ID NO: 13 and “INMWQ” as residues 7-11 of SEQ IDNO: 13.

FIG. 25: Amino acid sequence of gp120 of HIV-2 (SEQ ID NO: 30) comprisedin a fifth motif (for sequence of said motif see description hereinabove). FIG. 25 discloses “GGDMR” as residues 5-9 of SEQ ID NO: 12 and“GGDM” as residues 5-8 of SEQ ID NO: 12.

FIG. 26: Model of the tunnel structure in A and B, wherein in B thedarker shaded, horizontal section may be termed Site 1 of the tunnel andincludes external motif 4 and the lighter shaded horizontal section istermed Site 2 of the tunnel. C (Site 1) and D (Site 2) show models ofthe isolated sections of the tunnel and the adjacent external motif (forSite 1).

FIG. 27: A Alternate views of Site 1 and resolution of those parts ofsaid motifs 1, 2, 3 and 5 (partially making up the whole tunnel) and theexternal motif involved in forming Site 1.

FIG. 28: A Amino acid sequence of Site 1A (residues 81-142 of SEQ ID NO:2) (Bold amino acid residues (second motif) and sequence that is flankedby said bold residues). FIG. 28A discloses “VVSTQ” as residues 14-18 ofSEQ ID NO: 4. B Amino acid sequence of Site 1B (residues 198-259 of SEQID NO: 2) (Bold amino acid residues (third motif) and sequence that isflanked by said bold residues). FIG. 28B discloses “GDPEIVMHSFN” asresidues 3-13 of SEQ ID NO: 5 and “FFYC” as residues 18-21 of SEQ ID NO:5.

FIG. 29: A Amino acid sequence of Site 1C (residues 252-307 of SEQ IDNO: 2) (Bold amino acid residues (external motif) and sequence that isflanked by said bold residues). FIG. 29A discloses “IINMWQEVGKA” asresidues 6-16 of SEQ ID NO: 8 and “INMW” as residues 7-10 of SEQ ID NO:8. B Amino acid sequence of Site 1D (residues 310-344 of SEQ ID NO: 8)(Bold amino acid residues (fifth motif) and sequence that is flanked bysaid bold residues). FIG. 29B discloses “GGGDM” as residues 4-8 of SEQID NO: 7.

FIG. 30: A Alternate views of Site 2 and resolution of those parts ofsaid motifs 1, 2, 3 and 5 (partially making up the whole tunnel) and theexternal motif involved in forming Site 2.

FIG. 31: A Amino acid sequence of Site 2A (residues 6-74 of SEQ ID NO:2) (Bold amino acid residues (first motif, partially) and sequence thatis flanked by said bold residues). B Amino acid sequence of Site 2B(residues 253-307 of SEQ ID NO: 2) (Bold amino acid residues (externalmotif) and sequence that is flanked by said bold residues). FIG. 31Bdiscloses “INMWQ” as residues 7-11 of SEQ ID NO: 8.

FIG. 32: Amino acid sequence of Site 2X (residues 213-263 of SEQ ID NO:2) (Bold amino acid residues (third motif, partially) and sequence thatis flanked by said bold residues).

FIG. 33: A and B Alternate views of the relation of the parts 1A, B, Cand D and 2A, B and X in making up motifs 1, 2, 3 and 5 that partiallyform the tunnel within HIV and the external motif.

FIG. 34: A Model of compound T0520-5895 interacting with the motifs 1,2, 3, 5 and the external motif. B Alternate view of compound T0520-5895interacting with the motifs 1, 2, 3, 5 and the external motif.

FIG. 35: A Model of compound T5375934 interacting with the motifs 1, 2,3, 5 and the external motif and alternate view (B). C Model of compoundT0520-5895 interacting with the motifs 1, 2, 3, 5 and the external motifand its relation to Site 2. D Model of compound T0520-5895 interactingwith the motifs 1, 2, 3, 5 and the external motif and its relation toSite 1 and 2.

FIG. 36: ELISA Assay. A sandwich assay was developed to screen forcompounds that inhibit the binding of a4b7 to gp120. Without inhibitors,gp120 and a4b7 bind and this complex can be identified by addition of amouse anti-gp120 mAb (step 3). This a4b7·gp120·anti-gp120 mAb complex isthen sequestered on the bottom surface of a 96 welled-plate coated withan anti-mouse secondary mAb (step 4). The complex is then detected byaddition of a biotinylated primary mAb against a4b7 (step 5) anddetected using an HRP-avidin (step 6). A positive binding event isidentified by the presence of HPR activity within each well. Theaddition of an inhibitor (step 2) blocks the binding between gp120 anda4b7 leading to a lack or reduction of HRP activity as shown in steps2-5 at the bottom of the figure.

FIG. 37: Activity of a panel of inhibitors in blocking the binding ofgp120 to a4b7. (a) The left chart depicts data collected in example 3using ELISA assay. (b) The right chart depicts data collected byscreening cell extracts. Bold letters used for a compound denote activecompounds. The remaining compounds are weak or non-inhibitors All assayswere repeated six times (three times for each anti-gp120 mAb used in thecase of the ELISA data) and an average was presented. All values wereobtained within 5% deviation.

THE EXAMPLES ILLUSTRATE THE INVENTION Example 1 Binding Site of gp120.Model Validation and Pharmacophore Hypothesis

A 3D structure of gp120 protein interacting with CD4 is presented inFIG. 3 (1G9N from the Protein Data Bank (Wang et al., J. Med. Chem.48(12):4111, 4119 (2005))). As can be seen, Phe43 of CD4 enters intohydrophobic pocket within gp120 structure. The pocket is naturallyresponsible for specific recognition of the Phe43 residue of CD4. Thesmall size of the binding pocket is a significant obstacle forsuccessful docking of potential ligands. Since the protein structure inthe PDB Data Bank is only a single snapshot of the actual proteinconformational space, docking of any reasonably large or flexiblemolecule may fail and the correct orientation of ligand in the bindingsite may not be found. In what follows we outline a docking strategydesigned to overcome this difficulty.

Docking Procedure

Several 3D structures of gp120 protein from RSCB Protein Data Bank wereselected for docking (1G9M, 1G9N, 2B4C and 1GC1). To obtain more proteinconformations Molecular Dynamics simulation was performed starting fromthe 1G9M structure. The active sites volume in the protein conformationsalong the trajectory generated by the Molecular Dynamics was followedaiming to identify the conformations with largest binding pocket volume(see FIG. 5B).

Finally ˜60 protein conformations, both taken from PDB Data Bank andthose generated by Molecular Dynamics and corresponding to the largestvolumes of the binding sites, were selected and prepared for docking.Such a large number of different protein conformations used in thedocking study represents a reasonable portion of the proteinconformational ensemble, rather than a single snapshot made available ina ray experiment. Docking of a specific ligand to a single proteinstructure may not succeed; however docking of the same small molecule tomultiple conformations usually helps to identify the right binding mode.If a sufficiently large number of the protein conformations is used, themaximal value of the predicted binding affinity provides a goodindication of the actual ligand activity. This approach has been provedto be useful in various docking studies.

All small gp120 inhibitors with known chemical structure andexperimentally determined biological activity where included in theresearch. Both the proteins and small molecules typization, and insilico screening were carried out by the molecular processing anddocking tools taken from the QUANTUM drug discovery software suite[QUANTUM]. The software predicts the binding affinities of smallmolecules to resolved protein targets using a set of principles based onmolecular simulations with an advanced continuous water model (P. O.Fedichev and L. I. Men′shikov. Long-range order and interactions ofmacroscopic objects in polar liquids, 2006.). The approach provides thelogarithmic values of the binding constant, pKd (−1 gKd), with theaccuracy of about one pKd unit.

Docking of the molecules was performed on refined gp120 proteinstructures. The docking box 12×18×20 covers all the residues importantfor ligand binding, see FIG. 1. After the docking all the ligand-proteincomplexes were sent to Molecular Dynamics calculations for refinement.

The Nature of the Binding Site

Molecular dynamics simulations provide an important insight into thenature of the gp120 protein ligand binding site. To visualize theconsequences of the protein thermal motion the volume of the dockingspace associated with certain binding areas of Site 1 (residues 368,370, 371, 427, 457, 357 of gp120) and of Site 2 (112, 113, 382, 426,125, 429, 433, 475 of gp120) was monitored, see FIG. 5B. As it is clearfrom the graphs, the volumes of the active sites associated with both ofthe residues sets undergo distinct low frequency oscillations on top ofthe random thermal noise. Such long period movements are oftenassociated with important structural changes.

A closer look at the structural transition reveals the opening of awater filled “tunnel” connecting the two pockets in the binding areas ofSites 1 and 2. The opening volume is fairly large and can accommodate anarrow and long ligand molecules, as shown in FIGS. 4 and 5. The tunnelis not present in any of the ray structures available in the PDB DataBank. The Figures show that apparently unrelated binding areas I and IIare indeed closely placed within the protein 3D structure. The overlapextends deeply inside the discovered tunnel, which means that thesuggested theory of the gp120 binding site provides a novel unifiedconcept bridging together different pieces of biological knowledge.

Moreover, as can be seen from our docking results, none of the publiclyaccessible gp120 PDB structures can be used for binding area Site 2binders docking.

Example 2 Profiling of Anti-HIV Activity

Two compounds were evaluated for their activity as inhibitor of HIVreplication on three different lab strains using the deCIPhR cellularformat. The two compounds were also tested for inhibition of replicationof the lab strain pNL4-3 in a virus-to-cell infection format. Theresults are reported in the table below as IC₅₀ values (concentration ofcompounds inhibiting 50% of viral replication).

Infec- tion Virus Tro- T0520- IC50 (nM) Type Strain pism T5379534 5895T20 EFV Cell to pNL-Bal R5 1538  15088* 10 5 Cell pNL-AD87 R5 766    250** 63 1 pNL4-3 X4 3199 >30000 76 9 Virus pNL4-3 X4 767 inactive 48 2 toCell *extrapolated values ** plateau of activity at ~70%

Results indicate that the two compounds T5379534 which corresponds tothe compound of formula (1) and T0520-5895 which corresponds to thecompound of formula (2) did show significant anti-HIV activity withoutsigns of cytotoxicity.

These two compounds were further tested on the replication of twoengineered viruses (“GIA” and “DIA” resistant to the fusion inhibitorT-20 (Enfuvirtide, Fuzeon). Results are reported in the table below asIC₅₀ values and as “resistance factor” (ratio between IC₅₀ value onmutant virus and IC₅₀ value on reference strain).

T5379534 T0520-5895 T20 EFV Virus Tro- IC50 IC50 IC50 IC50 Type pism(nM) Rf (nM) Rf (nM) Rf (nM) Rf pNL- R5 1538 1  15088* 1 10 1 5 1 Bal“G-I-A” R5 951 0.6  7597 0.5 127 12.7 1.7 0.3 “D-I-A” R5 20111 >30000 >40 2267 227 3 0.6 *extrapolated valuesB. Comments

The compounds T5379534 and T0520-5895 do show activity on referencestrains of HIV and variants resistant to T20.

C. Experimental Section

Preparation of Compounds

The compounds T5379534 (MW=436.15) and T0520-5895 (MW=491.18) wereprovided by Xenobe organization (Dr. James laClair) as aliquots of 1mg/mL solution. All other drugs used in the study were obtained from therespective pharmaceutical manufacturer.

Compound stocks were prepared as 1.5 mM solution in 100% DMSO and vialswere agitated for two hours at 50° C. Stock solutions were kept at −20°C., light protected as aliquots. Working dilutions (1/3 log) wereprepared extemporaneously by serial dilutions in H₂O/DMSO (95/5). Ten μLof working dilutions were then pipetted and directly tested for anti-HIVactivity in the 200 μL final volume of the deCIPhR cellular assayformat, yielding final concentrations of 7'500, 3'481, 1'616, 750, 348,162, 75, 35, 16, 8 and 3 nM.

1. Wild-Type Viruses

The activity of the compounds was profiled on three different virusesthat were chosen as reference for distinct co-receptor usage. Theircharacteristics are summarized for each virus in Table I.

TABLE 1 Characteristics of viruses selected for the study Virus pNL4-3pNL-Bal pNL-AD87 Origin USA USA USA Subtype B B B Co-receptor TropismCXCR4 CCR5 CCR5

1.1. pNL4-3

The proviral DNA pNL4-3 was obtained from the AIDS reagent center(Genbank accession number #AF324493). After transfection, pNL4-3produces a full-length infectious virus often used as reference for aB-Subtype, CXCR4-tropic strain (Adachi et al., 1986).

1.2. pNL-Bal

The DNA sequence coding for Vpu, Tat, Rev, Env, and Nef from theCCR5-tropic clone BAL2 (HIVBAL2A, Genbank accession number #M68894) wasintroduced by PCR-cloning into the reference proviral strain pNL4-3(Genbank accession number #AF324493), where it replaced thecorresponding region. Presence of insert was ascertained by restrictiondigest analysis and double sequencing on ABI 310 prism sequencer. Aftertransfection, pNL-BaL produces a full-length infectious virus often usedas reference for a B-Subtype, CCR5-tropic strain (Donaldson et al.,1994).

1.3. pNL-AD87

The DNA sequence coding for Vpu, Tat, Rev, Env, and Nef from theCCR5-tropic clone AD87 (HIV strain AD8, Genbank accession number#AF004394) was introduced by PCR-cloning into the reference proviralstrain pNL4-3 (Genbank accession number #AF324493), where it replacedthe corresponding region. Presence of insert was ascertained byrestriction digest analysis and double sequencing on ABI 310 prismsequencer. After transfection, pNL-AD87 produces a full-lengthinfectious virus often used as reference for a B-Subtype, CCR5-tropicstrain (Theodore et al., 1996).

2. Generation of ENV-Mutants

The two compounds were evaluated for possible cross-resistance to fusioninhibitors by testing activity on engineered proviruses carryingmutations described to lead to decreased susceptibility to T-20. Thethree amino-acids motif G36-I37-V38 (GIV) is part of a highly conservedregion within the Env gene (HR1) and was found to be mutated in patientsfailing T-20 treatment during Phase III clinical trials (Wei, X. et al.,Antimicrobial Agents and Chemotherapy, 46, 1896 (2002)).

Two distinct mutants with alterations within the gp41 sequence of theirEnv-gene were to be generated.

Mutant “G-I-A”: substitution from Valine to Alanine at amino-acidposition 38 of HIV gp41 (numbering according to reference lab strainpNL4-3, GenBank accession number #AF324493). This single mutation hasbeen described to lead to intermediate degree of resistance to thecompound T20 (Enfuvirtide, Fuzeon).

Mutant “D-I-A”: substitutions from Glycine to Aspartic Acid atamino-acid position 36 and from Valine to Alanine at amino-acid position38 of HIV gp41 (numbering according to reference lab strain pNL4-3,GenBank accession number #AF324493). This single mutation has beendescribed to lead to high degree of resistance to the compound T20(Enfuvirtide, Fuzeon).

The two mutants included in the present study were generated using anoverlapping-PCR strategy as schematically depicted in FIG. 6.

A first PCR step was performed using purified proviral DNA of pNL-Bal astemplate along with either the primer pair E1_F/mut1_R or the primerpair mut_1F/E2_R. Oligonucleotidic primers were as follows:

-   -   E1_F: forward primer corresponding to the sequence located        downstream from V2 (Nt 6942, numbering according to HxB2); this        primer contains the sequence for a restriction site of        restriction endonuclease E1;    -   mut1_R: this reverse primer containing the targeted mutation(s)        was designed to have at least 12-15 nucleotides complementary to        the 3′end of primer mut1_F (see annex);    -   mut1_F: this forward primer containing the targeted mutation(s)        was designed to have at least 12-15 nucleotides complementary to        the 3′end of primer mut1_R (see annex);    -   E2_R: reverse primer corresponding to the sequence located near        the middle of the gp41 sequence (Nt 8043, numbering according to        HxB2); it also contains the sequence of a restriction site for        restriction endonuclease E2.

All PCR-products generated by this first step were gel-purified,quantified using a nanodrop densitometer and combined at a 1:1 ratio. Anelongation step was then performed through 5 cycles in the absence ofprimers; primers E1_F and E2_R were then added, and a second step PCRreaction was performed.

Parameters of PCR reactions were as follows:

1^(st) Step PCR:

 2 min denaturation/enzyme activation at 98° C. 10 sec denaturation at98° C. | 10 sec annealing at 55° C. | 15 sec extension at 72° C. | → 25cycles 10 min final extension at 72° C.Intermediate Elongation:

 2 min denaturation/enzyme activation at 98° C. 10 sec denaturation at98° C. | 10 sec annealing at 55° C. | 20 sec extension at 72° C. | → 5cycles2^(nd) Step PCR

 2 min denaturation/enzyme activation at 98° C. 10 sec denaturation at98° C. | 10 sec annealing at 55° C. | 30 sec extension at 72° C. | → 30cycles 10 min final extension at 72° C.

Next, 1.1 Kb PCR products were gel-purified through electrophoresis on a0.8% agarose gel/Tris-Borate EDTA. Fragments were excised and elutedusing ion exchange columns (Macherey-Nagel). Purified DNA fragments weresubsequently digested with restriction enzymes E1 and E2 and ligatedinto a cloning cassette previously prepared by digestion withrestriction endonucleases E1 and E2. Ligated recombinant proviral DNAwas transformed into E. coli bacteria (HB 110/λ) and grown on LB-Agarcontaining 200 μg/mL ampicillin as resistance marker. Single colonieswere picked and grown overnight in LB medium containing 200 μg/mLampicillin. Amplified plasmid was prepared using a miniprep extractionkit (Macherey-Nagel) and sequenced using a dideoxy sequencing kit(Applied Biosystems).

3. Infection Experiments

3.1 Principle of Decipher

Principle of Dual-Enhancement of Cell-Infection to Phenotype Resistance(deCIPhR™): deCIPhR test is a proprietary assay system developed byInPheno AG (FIG. 7):

Fully infectious HIV-1 is produced from a proviral reference DNAreflecting a reference HIV-1 virus. Recombinants carrying an exchangedspecific-sequence (e.g. mutants resistant to existing HIV-1 inhibitorsor sequences issued from clinical situations) can easily be engineeredby PCR-based amplification/ligation (see paragraph 3). The unique testformat allows 3 to 5 rounds of viral replication and permits a dynamicparallel read-out of viral fitness and resistance to the respectivedrugs under investigation. Colorimetric readout (405 nm) is translatedin percent viral inhibition through normalization with positive andnegative control wells included in each 96 well-plate.

The format of deCIPhR includes characteristics essential for optimalassessment of parameters of viral replication:

-   -   Generation of restriction-recombinant virus is more rapid and        efficient, and position-defined and thus proves to be superior        to pseudotypes;    -   Cell to cell infection most closely mimics viral behavior within        the host;    -   A replicative system bests reflects viral dynamics.

Each mutated proviral DNA is transfected into a human epitheloid cellline using lipofectant agent (lipofectamine 2000, Invitrogen) followingthe manufacturer's instructions. Cell to cell spread and replication ofrecombinant viruses is allowed for but also restricted to a period offour days in the absence or presence of specific drugs by co-culturewith a second cell line expressing both chemokine receptors CXCR4 andCCR5. Finally, culture supernatants are transferred to a third reportercell line also expressing both kinds of co-receptors. The infection isincubated for another 48 hours period, after which cells are fixed(formaldehyde/glutaraldehyde) prior to incubation with a chromogenicsubstrate for beta-galactosidase, typicallyortho-nitrophenyl-galactopyranoside.

3.2 Principle of Virus to Cell Infection (FIG. 8)

Fully infectious HIV-1 is produced from proviral reference DNAreflecting a reference wild-type HIV-1 virus (pNL4-3, Genbank#AF003888). The unique test format allows direct viral infection andreplication and permits a dynamic parallel read-out of viral fitness andresistance to the respective drugs under investigation.

Format for virus to cell infection: Briefly, the assay was performed in6 well tissue culture plates and included 50'000 HIV-producing cells(HeLa) in a final volume of 2 mL DMEM medium (Gibco, Parsley, UK). After2 days of incubation, 44 of viral supernatant was added to 5'000pre-incubated HIV-reporter cells (HeLaCCR5) in a final volume of 2004 ofRPMI medium (Gibco, Parsley, UK) including 104 of substances to betested.

3.3 Data-Processing for Activity Determination

A range of twelve concentrations of each compound was tested intriplicates in the deCIPhR format on each mutated provirus. Thecolorimetric readout obtained at the end of the deCIPhR experiment(optical density at 405 nm) was translated into a percentage of viralinhibition as function of compound concentration and processed by astatistical curve fitting software (XLfit v 4.0.1, IDbusiness solutions,Guilford, UK) yielding the selection of a best-modeled curve accordingto the following equation (equation 205: one-site pharmacologicaldose-response):

$y_{1} = {{\int_{A}^{B}A} + \frac{B - A}{1 + \left( \frac{C_{1}}{x_{1}} \right)^{D_{1}}}}$

Where:

-   -   y₁ is the modeled effect (% viral inhibition) of drug 1 at        concentration x1    -   x₁ is the concentration of drug 1 yielding the modeled effect y1    -   A is a constant fixed to 0    -   B is a constant fixed to 100    -   C₁ is the concentration required to obtain 50% of the modeled        effect (IC50) for drug 1    -   D₁ is the slope of the modeled curve (Hill's coefficient) for        drug 1.

This algorithm used integral calculations to model inhibition curves foreach compound, taking into account values of the triplicates and theobserved standard deviations and allows to directly extract IC₅₀ andIC₉₀ values.

3.4 Replication Capacity Determination

The cellular system deCIPhR allows the rapid evaluation of drug activityon any variant/mutant of HIV-1. Viruses produced from a proviral DNA areallowed to replicate through 3 to 5 cycles in the cell system therebyallowing to observe dynamics of the viral replication. The enzymaticreporter read-out has been demonstrated to be strictly proportional tothe production of viral particles as measured by quantitative RT-PCR ofviral RNA, Reverse-transcriptase activity, and the concentration of p24antigen in the culture supernatant. Mutated viruses engineered in thepresent study differ by mutations in Env gene (HR1). The remaining partof the proviral genomic DNA outside this region stems from the clonalreference provirus NL-Bal thus providing an isogenic background.Therefore, whereas the relative susceptibility of mutated viruses tofusion inhibitors such as the two test compounds is not known a-priori,the susceptibility to Reverse transcriptase inhibitors (RTIs) isexpected to be identical. Concentrations of RTIs inhibiting 100% ofviral growth without signs of cytotoxicity (hereafter termed IC₁₀₀) havepreviously been determined. The viral “fitness” or “replicationcapacity” was measured as a percentage of the respective replicationcapacity of the reference virus, calculated as follows:

${{RC}(\%)} = {\frac{{{Readout}_{100\%}({sample})} - {{Readout}_{0\%}({sample})}}{{{Readout}_{100\%}({ref})} - {{Readout}_{0\%}({ref})}} \times 100}$

Whereby:

-   -   RC (%)=Replication capacity of the recombinant virus under        investigation    -   Readout_(100%)(sample)=enzymatic reporter readout of the mutated        virus under investigation (average of 6 values) in the absence        of inhibitor.    -   Readout_(0%)(sample)=enzymatic reporter readout of the mutated        virus under investigation (average of 6 values) in the presence        of IC100 concentration of 10 μM Efavirenz as RTI.    -   Readout_(100%)(ref)=enzymatic reporter readout of the reference        virus (pNL-Bal) (average of 6 values) in the absence of        inhibitor.    -   Readout_(0%)(ref)=enzymatic reporter readout of reference virus        (pNL-Bal) (average of 6 values) in the presence of IC100        concentration of 10 μM Efavirenz as RTI.        4. Results        4.1 Activity of Two Compounds on Replication of Wild-Type        Viruses

The two compounds under investigation were tested along with referencecompounds T20 and EFV on three wild-type viruses (pNL4-3, green curves;pNL-Bal, blue curves and pNL-AD87, orange curves) using the deCIPhRcellular system (see paragraph 4.1). The results of the inhibition ofHIV-replication by the four compounds are reported in FIG. 9 asinhibition curves where the percentage of inhibition of HIV-replicationis plotted as a function of compound concentration (described inparagraph 4). Tables below the graphs report the inhibition observed ateach concentration as well as derived parameters for compounds underinvestigation and also for reference compounds.

Of note is that no signs of cytotoxicity was microscopically observedfor the all compounds at tested concentrations.

Next, the two compounds were assessed for inhibition of viralreplication in a virus-to-cell infection format along with referencecompounds T20 and EFV. In this experiment only pNL4-3 reference HIVstrain was used.

Graphs in FIG. 10 show the inhibition curves as percent of virusinhibition as a function of increasing concentrations of each compound.The table in FIG. 10 reports the calculated parameters of HIV inhibition(IC50, IC90 and Hill's coefficient).

Of note is that signs of cytotoxicity were microscopically observed forthe highest concentration of compounds T5379534 and T0520-5895.

4.1 Activity of the Compounds on Replication of Engineered VirusesResistant to the Fusion Inhibitor T20 (Fusion Inhibitor)

In this subsection of the study the compounds T5379534 and T0520-5895were tested on the mutated viruses “G-I-A” and “D-I-A”.

4.2.1 Activity of T5379534 and T0520-5895 on “G-I-A” Mutant

The two compounds under investigation were tested along with referencecompounds T20 and EFV on the mutated virus “G-I-A” in which the Valineat position 38 of gp41 from pNL-Bal is substituted with an Alanine. Theresults of the inhibition of HIV-replication by the four compounds arereported in FIG. 11 as inhibition curves where the percentage ofinhibition of HIV-replication is plotted as a function of compoundconcentration (described in paragraph 4). Tables below the graphs reportthe inhibition observed at each concentration as well as derivedparameters for compounds under investigation and also for referencecompounds.

4.2.2 Activity of T5379534 and T0520-5895 on “D-I-A” Mutant

The two compounds under investigation were tested along with referencecompounds T20 and EFV on the mutated virus “D-I-A” in which the Glycineat position 36 of gp41 from pNL-Bal is substituted with an Aspartic Acidand the Valine at position 38 with an Alanine. The results of theinhibition of HIV-replication by the four compounds are reported in FIG.12 as inhibition curves where the percentage of inhibition ofHIV-replication is plotted as a function of compound concentration(described in paragraph 4). Tables below the graphs report theinhibition observed at each concentration as well as derived parametersfor compounds under investigation and also for reference compounds.

4.3 Calculation of Resistance Factor

In order to be able to assess possible cross-resistance of mutants tothe two evaluated compounds a Resistance factor was calculated for eachcompound on each mutant following the equation:

${{Resistance}\mspace{14mu}{factor}_{x,m}} = \frac{{IC}_{{50x},m}}{{IC}_{{50x},{wt}}}$

Where:

-   -   Resistance factor_(x,m): Resistance factor of compound x on        mutant m    -   IC_(50x,m): concentration of compound x necessary to inhibit 50%        of viral replication of mutant m    -   IC_(50x,wt): concentration of compound x necessary to inhibit        50% of viral replication of wild-type pNL-Bal

Resistance factors were calculated for all compounds and reported as“IC50-fold increase” in table II:

TABLE II Resistance factor values calculated for each compound on eachmutant. Resistance factor Cpds (IC_(50 mut)/IC_(50 wt)) IC₅₀ foldincrease mutant T5379534 T0520-5895 T20 EFV “G-I-A” 1 0.5 13 0.3 “D-I-A”1 >40 227 0.5

REFERENCES OF EXAMPLE 2

-   Adachi A, et al. (1986) Production of acquired immunodeficiency    syndrome-associated retrovirus in human and nonhuman cells    transfected with an infectious molecular clone. J. Virol. 59(2):    284-291.-   Donaldson Y K, et al. (1994) In vivo distribution and cytophatology    of variants of human immunodeficiency virus type 1 showing    restricted sequence variability in the V3 loop. J. Virol. 68(9):    5991-6005.-   Theodore T S, et al. (1996) Construction and characterization of a    stable full-length macrophage-tropic HIV type 1 molecular clone that    directs the production of high titers of progeny virions. AIDS Res    Hum Retroviruses. 12(3):191-194.-   Wei X., et al. (2002) Emergence of resistant human immunodeficiency    virus type 1 in patients receiving fusion inhibitor (T-20)    monotherapy. Antimicrob Agents Chemother., 46 (6), 1896-1905.

Example 3 Gp120 Binding to a4b7

Studies were conducted to study the binding of gp120 to the integrinα4β7 (or a4b7; alpha4 beta7 as used herein above) and to develop ahigh-throughput assay to screen for compounds that interfere with thebinding of HIV associated glycoprotein gp120 and the integrin α4β7.

A. Materials.

General methods were used to obtain the necessary cells, proteins,antibodies and reagents. When possible, commercial materials were usedand the vendor and product number are designated.

Antibodies.

A goat HIV1 anti-gp120 polyclonal antibody (ab21179) was obtained fromAbcam. Mouse anti-gp120 monoclonal antibodies (mAbs) were obtained fromAbcam [HIV1 gp120] (ab13411) and Prospec [HIV-1 gp120] (ANT-151). MousemAbs were used in the ELISA assay and the goat polyclonal antibody wasused for protein production. A rat mAb against integrin α4β7 [DATK32](ab25329) was obtained from Abcam Inc. In addition, mouse antibodiesagainst the human integrin α4 [44H6] (ab220) and human integrin β7 [8G2](mca5238Z) were obtained from Abcam Inc. and AbD Serotec Inc.,respectively, and used for protein purification. The integrin α4β7 mAbwas also labeled with biotin using EZ-Link Sulfo-NHS biotinylation kit(21425) from ThermoScientific using the procedures described in themanufacturers protocol.

Gp120 Protein.

HIV-1 gp120 plasmid was also obtained containing the gp120 gene from anM cell-tropic HIV-1 ADA strain. The recombinant envelope gp120glycoprotein was also previously produced in the Baculovirus ExpressionSystem (Invitrogen) on a hollow-fiber filter cell device (Filter CellSystems Inc) in Sf9 cells (Orbigen Inc.). Crude recombinant envelopegp120 glycoprotein was purified by prep-fast protein liquidchromatography (FPLC). This method was used to prepare the gp120 proteinfor prior studies. Reapplication of the method delivered 4.2 mg of gp120protein with a purity of over 95% purity by SDS PAGE analysis using aSilverQuest kit (Invitrogen) for detection.

Alpha4 Beta7 (a4(37) or LPAM-1 Protein.

Recombinant human α4β7 integrin was purchased from R&D Systems (CatalogNumber: 5397-A3). Larger quantities of the α4β7 integrin were by inhouse. Recombinant expression of both subunits α4 and β7 wasaccomplished by preparation plasmids containing the α4 (proteinaccession # P13612) and β7 (protein accession # P26010) both containinga C-terminal 6×His tag (SEQ ID NO: 25). Both proteins were expressed inCHO cells using conventional methods and were purified to >98% purity(SDS PAGE analysis) by sequential His-tag purification on NTA-agarosefollowed by repetitive size exclusion purification using a SephadexG-200 column. The 6×His tags (SEQ ID NO: 25) were removed prior to sizeexclusion purification. The purity of each subunit was evaluated bySDS-PAGE analysis and both subunits were purified to over 98% purityusing a SilverQuest kit (Invitrogen) for detection. The α4β7 integrinwas reconstituted was prepared by incubation of a 1:1 mixture of the α4and β7 subunits followed by size exclusion purification by three passeson a Sephadex G-200 column. An anti-α4β7 mAb was used to identify thefractions containing the α4β7 integrin. This method was used to provide12.5 mg of the α4β7 integrin with greater than 96% purity. The activityof the α4β7 integrin was determined by using the methods established byR&D Systems Inc., as given by measuring the ability of the immobilizedα4β7 integrin to support the adhesion of VCAM-1 transfected Chinesehamster ovary (CHO) cells. When 5×10⁴ cells per well are added torhIntegrin α4β7 coated plates (10 μg/mL, 100 μL/well), between 60-80%will adhered in 1 h at 37° C. This procedure is described in the productcatalog for the α4β7 integrin (R&D Systems Inc.). All assays wereconducted with protein produced in our laboratories and was checked oncein triplicate against the commercial protein.

Reagents.

HRP-NeutrAvidin (21124) from ThermoScientific and QuantaBlu FluorogenicPeroxidase Substrate (15169) from ThermoScientific were used to developthe ELISA assays. All compounds were provided and stocked at 10 mg/mL inDMSO and stored at −80° C. until used. Buffers were all prepared assterile media and were stored for less than 24 h. All other reagents,plates, or devices are noted as used.

C. ELISA Analysis.

It was previously demonstrated that co-immunoprecipitation analyses asanalyzed via western blots were a viable means to evaluate the bindingof gp120 to α4β7 in human cell lysates. This method was advanced into anELISA format and applied to screen the number of compounds providedwithin the research period.

It was determined that the direction of the assay was not critical andantibodies can be used against both gp120 and the α4β7 integrins in anyorder. Based on these studies, an optimized ELISA assay as outlined inFIG. 36 was designed.

C.1. Assay Development.

The studies began by screening for the optimal protein concentrationsfor the method. The studies were conducted in goat anti-mouse IgG coatedblack React-Bind 96 welled-plates (R&D Biosystems), referred to hereinas the anti-mouse IgG plate. We prepared twelve stock solutionscontaining a 1:1 stoichiometric mixture of gp120 and α4β7 integrin inPBS at pH 7.2 as given by 0 μM or control, 0.001 μM, 0.01 μM, 0.01 μM,0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM and 25 μM in protein(step 1, FIG. 36). A 200 μL aliquot of each stock solutions was thenloaded across a 96 welled plate and treated either with 20 μL of PBS pH7.2 (control) or 20 μL of a 100 μM stock solution of repandusinic acidin PBS pH 7.2 containing 1% DMSO. Three repetitions were run for boththe control and positive or repandusinic acid treated experiments. Thefinal concentration of repandusinic acid in each positive well was 10μM. The plate was incubated for 4 h at 4° C. on a plate mixer at a speedthat created a vortex in each well.

During this time, repandusinic binds to blocks the formation of theα4β7·gp120 complex (step 2, FIG. 36). This process provided a platecontaining the antigens, or so called antigen plate.

In parallel, the anti-mouse IgG plate was washed 3 times with 200 μL ofwash buffer (PBS pH 7.2. containing 0.05% Tween 20) and treated with 100μL of a 0.5 μg/mL stock of the mouse anti-gp120 mAb in PBS pH 7.2. TwomAbs were tested (see Materials Section above). Data was reported usinga combination from three repetitions from each mAb, affording an averageover six experiments, as indicated by step 3 (FIG. 36). This processdelivered the binding plate. After incubating the plate for 1 h at 23°C. on a plate mixer at a speed that created a vortex in each well, eachwell drained by aspiration and washed three times with 200 μL of washbuffer. The contents of the antigen plate (above) were transferred tothe complementary well son the binding plate. The binding plate wasshaken for 1 h at 23° C. on a plate mixer at a speed that created avortex in each well, as indicated by step 4 (FIG. 36).

Each well of the binding plate was aspirated and rinsed three times with200 μL of wash buffer. The wells were charged with 100 μL of a 0.1 μg/mLof the rat anti α4β7 mAb and the plate was shaken for 1 h at 37° C.(step 5, FIG. 1). This process was then repeated using 100 μL of 0.2μg/mL solution of the HRP-conjugated strepavidin (step 6, FIG. 36). TheHRP activity was developed using QuantaBlu fluorogenic peroxidisesubstrate (ThermoScientific) and evaluated on a HTS7000 plate reader(Perkin Elmer). Using this method it was determined that the idealconcentration of gp120 and α4β7 was 0.5-1.0 μM.

C.2. Optimization.

Then the assay was exhaustively tested by screening the inhibition ofthe binding of gp120 and α4β7 by repandusinic acid. Stock solutions ofrepandusinic acid (10× stock solutions) were prepared at 0 μM orcontrol, 0.01 μM, 0.1 μM, 0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, 25 μM, 50μM, 100 μM and 250 μM). Using this gradient, we were able to identifythe following optimized protocol.

Step 1: Prepare the Antigen Plate

-   -   a. Prepare a PBS stock solution containing 1 μM gp120 and 1 μM        α4β7    -   b. Add a 200 μL aliquot to each well of the antigen plate.

Step 2: Add the Inhibitor.

-   -   a. Add 20 μL of a 10× stock of the inhibitor in PBS pH 7.2        containing 1% DMSO    -   b. Incubate at 4° C. for 6 h with shaking. This delivers the        antigen plate.

Step 3: Prepare the Binding Plate.

-   -   a. Aspirate each well of the binding plate    -   b. Wash three times with 200 μL of wash buffer (PBS pH 7.2.        containing 0.05% Tween 20).    -   c. Add 100 μL of a 0.5 μg/mL stock of the mouse anti-gp120 mAb        in PBS pH 7.2    -   d. Shake for 1 h at 23° C.

Step 4: Sequester the Gp120·α4β7 Complex.

-   -   a. Aspirate each well of the binding plate.    -   b. Wash three times with 200 μL of wash buffer.    -   c. Transfer the contents of the antigen plate to the        corresponding wells in the binding plate.    -   d. Shake for 1 h at 23° C.

Step 5: Develop the Binding Plate.

-   -   a. Aspirate each well of the binding plate.    -   b. Wash three times with 200 μL of wash buffer.    -   c. Add 100 μL of a 0.1 μg/mL of the rat anti-α4β7 mAb.    -   d. Shake for 1 h at 37° C.    -   e. Aspirate each well of the binding plate.    -   f. Wash three times with 200 μL of wash buffer.    -   g. Add 100 μL of 0.2 μg/mL solution of the HRP-conjugated        strepavidin    -   h. Shake for 1 h at 37° C.    -   i. Aspirate each well of the binding plate.    -   j. Wash three times with 200 μL of wash buffer.    -   k. Develop using QuantaBlu fluorogenic peroxidase substrate        (ThermoScientific)    -   l. Evaluate the fluorescence output on a HTS7000 plate reader        (Perkin Elmer).

Outcome: Repandusinic acid A inhibited the binding of gp120 to α4β7 withan activity of 1.2±0.1 μM using the described an ELISA assay. Thisproved better than that obtained from use of the cell extracts 9.08 μMas determined in previous studies. The inhibition of the binding ofgp120 and α4β7 can be serialized and conducted in a 96 welled plateformat.

Application of the Gp120 and Integrin α4β7 Association Assay

The ELISA assay was applied to screen the panel of compounds to furthercharacterize their activity against the binding of HIV associatedglycoprotein gp120 and the integrin α4β7.

D. Implementation.

The five-step assay developed was applied to screen the panel ofcompounds provided. These materials were stored at −80° C. over theresearch period and were shown to be stable and retain purity by LC/MSanalysis prior to use. The experiments were run in triplicate using twoantibodies against gp120. The data was compiled and plotted (FIG. 37a )and compared against the results with less accuracy obtained in ourprior studies (FIG. 37b ). Only modest changes were observed between thein vitro studies conducted herein (FIG. 37a ) and those conducted oncell lysates (FIG. 37b ). Results obtained are described in detail inthe figure legend to FIG. 37 herein above.

Example 4 Profiling of Compounds Interacting with the Tunnel in ThreeHIV Strains

Preparation of Compounds

4 compounds namely, A03, G03, H04 and C05 identified as interacting withamino acid residues comprised in 6 motifs forming a tunnel within gp/20of HIV in a previous screen were tested against 3 HIV viruses expressingdifferent envelopes.

Compounds stocks were prepared as 20 mM solutions in 100% DMSO andstored light protected at −20° C. Because of solubility issues, anintermediate dilution to 5 mM in 100% DMSO was carried out for compoundA03 and to 2 mM for compounds G03, H04 and C05. Working solutions weremade just prior to use by serial dilutions in Phosphate-Buffered Saline(PBS)/DMSO (90/10) and added directly to the cell cultures (200 μL finalculture volume) to yield final concentrations of 25'000, 11'604, 5'386,2'500, 1'160, 539, 250, 116, 54 and 25 for compound A03 and 10'000,4'642, 2'155, 1'000, 464, 215, 100, 46 and 22 nM for the three othercompounds under investigation.

The fusion inhibitor T20 (Fuzeon, Roche & Trimeris), was used aspositive control. Viruses

The antiviral activity of the test substances was profiled on threeviruses that were selected according to their envelope sequence. Thecharacteristics of these viruses are summarised in Table III.

TABLE III Characteristics of viruses selected for the study Virus NL4-3Clone 896 Clone 94UG114 Origin USA USA Uganda Subtype B B D Co-receptorCXCR4 CXCR4 CXCR4 Tropism

The proviral DNA pNL4-3 was obtained from the AIDS reagent centre(Genbank accession number #AF324493). After transfection, pNL4-3produces a full-length infectious virus, NL4-3, often used as referencefor CXCR4-tropic B-Subtype strain (Adachi et al., 1986).

The proviral DNA p896 was obtained from the AIDS reagent centre (Genbankaccession number #U39362). After transfection of the proviral plasmid, afull-length infectious B-Subtype virus is produced (Collman et al.,1992).

The proviral DNA p94UG114 was obtained from the AIDS reagent centre(Genbank accession number #U88824). After transfection of the proviralplasmid p94UG114, a full-length infectious D-Subtype virus is produced(Gao et al., 1998).

Infection Experiment

Antiviral activity was determined according to the deCIPhR™ testdescribed herein above (cf. FIG. 7).

Data Processing for Activity Determination

A range of nine concentrations of each test substance provided by KFLPBiotech was tested in triplicate in the deCIPhR assay described above.The colorimetric readout obtained at the end of the experiments (opticaldensity at 405 nm) was translated into percent viral inhibition. To thisend, each plate included control wells for 100% readout (diluent only,mock treated) and 0% readout i.e., wells containing 300 nM of Efavirenz,a reference inhibitor, which reflects a concentration known tocompletely inhibit viral replication in vitro. The readout of each wellwas then transformed to % viral inhibition using the formula:% viral inhibition X=100−(((readout X−readout 0%)/(readout 100%−readout0%))×100)Where:

-   -   readout X=OD405 nm of well containing ‘X’    -   readout 100%=average of OD₄₀₅ nm of “100% readout” wells    -   readout 0%=average of OD₄₀₅ nm of “0% readout” wells

Using these transformed data, curve fitting was then performed with thehelp of XLfit software (version 4.3.2, IDbusiness solutions, Guilford,UK) yielding the selection of a best-modelled curve according to thefollowing equation (equation 205: one-site pharmacologicaldose-response):

$y_{1} = {{\int_{B}^{A}A} + \frac{B - A}{1 + \left( \frac{C_{1}}{x_{1}} \right)^{D_{1}}}}$

Where:

-   -   y1 is the modelled effect (% viral inhibition) of drug 1 at        concentration x1    -   x1 is the concentration of drug 1 yielding the modelled effect        y1    -   A is a constant fixed to 0    -   B is a constant fixed to 100    -   C1 is the concentration required to obtain 50% of the modelled        effect (EC₅₀) for drug 1    -   D1 is the slope of the modelled curve (Hill's coefficient) for        drug 1.

This algorithm uses integral calculations to model inhibition curves foreach compound, taking into account values of the triplicates and theobserved standard deviations and allows to directly extract EC50 andEC90 values.

Results

In the below Table IV there is a summary of deCIPhR™ assay resultsdisplayed as effective concentration of the tested compounds inhibitingNL4-3, 896 and 94UG114 replication to 50% and 90% (EC₅₀ and EC₉₀). Dueto the interaction with highly conserved amino acid residues forming ahighly conserved structure within gp120, i.e. the tunnel describedherein, all tested compounds exhibit antiviral activity in the differentHIV strains.

TABLE IV EC50 (μM) EC90(μM) A03 NL4-3 8.1 29.2* 896 9.1 34.3* 94UG1149.7 145.4* G03 NL4-3 3 8.6 896 4.6 10.5 94UG114 2.5 8.4 H04 NL4-3 2.713.3* 896 4.6 11.2* 94UG114 4 4.4 C05 NL4-3 10.2 55.7* 896 10.2 63.7*94UG114 3.8 5.1 *extrapolated values

REFERENCES OF EXAMPLE 4

-   Adachi A, et al. (1986) Production of acquired immunodeficiency    syndrome-associated retrovirus in human and nonhuman cells    transfected with an infectious molecular clone. J. Virol. 59(2):    284-291.-   Collman R, et al. (1992) An infectious molecular clone of an unusual    macrophage-tropic and highly cytophatic strain of human    immunodeficiency virus type 1. J. Virol. 66(12): 7517-7521.-   Gao F, et al. (1998) A comprehensive panel of near-full-length    clones and reference sequences for non-subtype B isolates of human    immunodeficiency virus type 1. J. Virol. 72(7): 5680-5698.

The invention claimed is:
 1. A method of identifying an inhibitor of thebinding of human immunodeficiency virus (HIV) glycoprotein 120 (gp120)to a CD4-receptor or to the integrin alpha4 beta7 (a4b7), the methodcomprising: (a) bringing into contact a HIV gp120 or a peptidomimeticreflecting the three-dimensional structure of said gp120 and a testcompound; (b) determining whether said test compound interacts with atleast two amino acid residues found independently in each of three ofsix motifs, said three of six motifs selected from the group consistingof motif 1, motif 2 and motif 3, within the 3-dimensional structure ofsaid gp120 or within a peptidomimetic reflecting the 3-dimensionalstructure of said gp120, wherein a first motif of said six motifscomprises the amino acid sequence DIISLWDQSLKPCVKLT (SEQ. ID. NO. 3(HIV-1) or SEQ. ID. NO. 9 (HIV-2)), wherein a second motif of said sixmotifs comprises the amino acid sequence NVSTVQCTHGIRPVVSTQLLLNGSLAE(SEQ. ID. NO. 4 (HIV-1) or SEQ. ID. NO. 10 (HIV-2)) and wherein theinteraction with any amino acid residue of said second motif is with anyone or more of residues 14-18 of SEQ. ID. NO. 4 (HIV-1) or SEQ. ID. NO.10 (HIV-2); wherein a third motif of said six motifs comprises the aminoacid sequence SGGDPEIVMHSFNCGGEFFYCN (SEQ. ID. NO. 5 (HIV-1) or SEQ. ID.NO. 11 (HIV-2)); wherein a fourth motif of said six motifs comprises theamino acid sequence CPKISFEP (SEQ. ID. NO. 6 (HIV-1) or SEQ. ID. NO. 14(HIV-2)); wherein a fifth motif of said six motifs comprises the aminoacid sequence FRPGGGDMRDNWRSELYKYKVV (SEQ. ID. NO. 7 (HIV-1)); andwherein a sixth motif of said six motifs comprises the amino acidsequence CSS, said gp120 of HIV comprising or consisting of: (i) thesequence of SEQ ID NO: 2; (ii) the sequence encoded by the sequence ofSEQ ID NO: 1; (iii) a sequence being at least 50% identical to thesequence of SEQ ID NO: 2 or to the sequence encoded by the sequence ofSEQ ID NO: 1; or (iv) a sequence encoded by a sequence being at least50% identical to the sequence of SEQ ID NO: 1, wherein said sequence of(iii) or (iv) comprises or encodes said motifs or variants thereof; and(c) identifying those compounds which interact with at least two aminoacid residues found independently in any three motifs selected from thegroup consisting of motif 1, motif 2 and motif 3, in said six motifswithin the 3-dimensional structure of said gp120 in (b).
 2. The methodof claim 1, wherein said determining in step (b) is effected by X-raycrystallography and/or NMR spectroscopy.
 3. The method of claim 1,further comprising the step of (a′) (i) determining whether said testcompound forms a complex with said gp120; and/or (ii) determiningwhether said test compound modulates the activity and/or conformation ofsaid gp120; and/or (iii) determining the cytotoxicity of said testcompound; wherein step (a′) is to be effected after step (a) and priorto step (b), and wherein said determining in step (b) is performed withtest compounds determined to bind, to modulate, and/or to benon-cytotoxic in step (a′).
 4. The method of claim 3, wherein saidactivity is the capability of said gp120 to mediate viral entry of HIVinto mammalian cells.